Natural Polyphyllins (I, II, D, VI, VII) Reverses Cancer Through Apoptosis, Autophagy, Mitophagy, Inflammation, and Necroptosis
Received 3 December 2020
Accepted for publication 19 February 2021
Published 10 March 2021 Volume 2021:14 Pages 1821—1841
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Leo Jen-Liang Su
Bashir Ahmad,1,2 Yaser Gamallat,3 Muhammad Fiaz Khan,4 Syed Riaz Din,2 Muhammad Israr,1,5 Manzoor Ahmad,6 Naeem Tahir,2 Nasir Azam,2 Khalil Ur Rahman,2 Wang Xin,7 Wang Zexu,7 Peng Linjie,7 Pengyu Su,2 Wang Liang8
1Department of Biology, University of Haripur, KPK, I. R. Pakistan; 2College of Basic Medical Sciences, Dalian Medical University, Dalian, 116044, People’s Republic of China; 3Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou, People’s Republic of China; 4Zoology Department, Hazara University, Mansehra, I. R. Pakistan; 5Biochemistry and Molecular Biology, College of Life Science, Hebei Normal University, Hebei, People’s Republic of China; 6Department of Chemistry, Malakand University, Chakdara, KPK, I. R. Pakistan; 7Department of Biotechnology, College of Basic Medical Sciences, Dalian Medical University, Dalian, 116044, People’s Republic of China; 8Stem Cell Clinical Research Center, National Joint Engineering Laboratory, Regenerative Medicine Center, The First Affiliated Hospital of Dalian Medical, Dalian City, Liaoning Province, 116011, People’s Republic of China
Correspondence: Wang Liang; Bashir Ahmad Email [email protected]; [email protected]
Abstract: Cancer is the second leading cause of mortality worldwide. Conventional therapies, including surgery, radiation, and chemotherapy, have limited success because of secondary resistance. Therefore, safe, non-resistant, less toxic, and convenient drugs are urgently required. Natural products (NPs), primarily sourced from medicinal plants, are ideal for cancer treatment because of their low toxicity and high success. NPs cure cancer by regulating different pathways, such as PI3K/AKT/mTOR, ER stress, JNK, Wnt, STAT3, MAPKs, NF-kB, MEK-ERK, inflammation, oxidative stress, apoptosis, autophagy, mitophagy, and necroptosis. Among the NPs, steroid saponins, including polyphyllins (I, II, D, VI, and VII), have potent pharmacological, analgesic, and anticancer activities for the induction of cytotoxicity. Recent research has demonstrated that polyphyllins (PPs) possess potent effects against different cancers through apoptosis, autophagy, inflammation, and necroptosis. This review summarizes the available studies on PPs against cancer to provide a basis for future research.
Keywords: natural products, saponins, polyphyllins, apoptosis, autophagy, inflammation, necroptosis
Cancer is the second leading cause of mortality worldwide.1–4 A WHO report shows that cancer causes more deaths in the world compared with stroke and coronary heart diseases.5 Factors that increase cancer incidence include overpopulation and aging.6 Global epidemiologic and demographic transition signals indicate that cancer will increase in the next decade, especially in countries with low or middle income.7 A recent report has estimated that 18.1 million new cancer cases excluding non-melanoma skin cancer (17.0 million cases) and 9.6 million cancer-related deaths excluding non-melanoma skin cancer (9.5 million cases) were recorded in 2018.8 In both genders combined, lung cancer (11.6% of total reported cases) was more common, followed closely by female breast (11.6%), prostate (7.1%), and colorectal (6.1%) cancers. Lung cancer was also the leading cause of death (18.4% of the total reported cases), followed by colorectal cancer (9.2%), stomach (8.2%), and liver (8.2%) cancers.8
Available clinical treatment for cancer includes surgery, chemotherapy, and radiotherapy.9 Conventional therapies, including surgery, radiation, and chemotherapy, have limited success because of secondary resistance.1,10 Therefore, safe, non-resistant, less toxic, and convenient drugs are urgently required.1,10 NPs, primarily sourced from medicinal plants, are ideal for cancer treatment because of their low toxicity and high success.1,11,12 These NPs cure cancer by regulating different pathways, such as PI3K/AKT/mTOR, NF-kB, autophagy, MEK-ERK, inflammation, oxidative stress, and apoptosis.11
Paris (Melanthiaceae) consists of 29 species,13 many of which are used as traditional herbal medicines14 and distributed in Eastern Asia and Europe. Among 29 species of Paris,13 22 are found in China and its diversity center in China is South-west China.15 In China, extracts from Paris species are extensively used as herbal medicine.16 Paridis, the dry rhizome of Paris, is used as a main raw material in patent Chinese drugs, including “GongXue Ning,” “Jidesheng snake tablet,” and “Yunnan Baiyo”.17 For a thousand years, this herb has been used in traditional Chinese medicine for the treatment of various diseases, such as snakebite, parotitis, hemostasis, abscess, and fractures.17 Modern research explored its new pharmacological activities, including cytotoxic, hemolytic, antibiotic, spermicidal, styptovit analgesic, calming, immunoregulatory, anthelmintic, anti-inflammatory, and anticancer activities.18–22
Photochemical research found several active ingredients in the dried rhizome of Paris, including flavonoids, endophytic fungi, fatty acid ester, and steroid saponins, such as Polyphyllin I (PPI), Polyphyllin II (PPII), Polyphyllin VI (PPVI), and Polyphyllin VII (PPVII).17,21,23 Modern research revealed that the PPs have potential pharmacological, analgesic, and anticancer activities for the induction of cytotoxicity through apoptosis.24–26
PPI,15,27–31 PPII,15,27–31 PPVI,15,27–31 and PPVII15,27–32 have been isolated from 13 Paris species, including, P. polyphylla (ChongLou),32 P. caobangensis,15,27,28 P. cronquistii,15,27,28 P. cronquistii var. xichouensis,15,27,28 P. delavayi var. petiolata,15,27,28 P. fargesii,15,27,28 P. mairei,15,27,28 P. polyphylla,15,27–29 P. polyphylla var. alba,15,27,28 P. polyphylla var. chinensis,15,27,28 P. polyphylla var. younaninsis,15,27,28,30,31 P. vitenaminsis,15,27,28 P. axialis pseudothi,15,27,28 and P. polyphylla var.15,27,28 These studies reported that the amount of isolated compounds from the same species of different regions varies possibly because of climate changes.15,27 PPD,23,33–37 Paris-VII23,33,34 are derived from Rhizoma paridis,23,33,34 Paris polyphylla,37 P. forrestii.35,36 Rhizoma Paridis extracts also containing PPI, PPII, PPVI and PPVII.38
Among these PPs, PPI has a significant therapeutic effect on hepatocellular carcinoma, lung adenocarcinoma, and gastric cancer.39–41 PPII has different pharmacological activities, including hemolytic effect42 and anticancer (eg, against ovarian cancer).43 PPVI inhibits colon cancer by inducing apoptosis, PPVII is a potent compound for the treatment of cervical cancer by inhibiting the growth of Hela cells.44 PPD exerts a potent antiproliferative effect against different types of cancer cells, including HepG-2, MCF-7 and MDA-MB-231.25,45,46 These PPs exhibit an anticancer effect against different cancers through different mechanisms. This review summarizes the anticancer mechanisms of these PPs to provide basic knowledge for further studies.
Non-Mechanistic Studies of PPs in Different Cancers
This section discusses those studies in which PP mechanisms are not reported. PPD inhibits the growth of HMEC-1 cells and decreases angiogenesis by suppressing cell proliferation, tube formation, and migration in vitro.47 PPD induces apoptosis in HL-60, SMMC-7721, A-549, MCF-7, and SW480 cell lines.35 PPD inhibits the proliferation of ovarian cancer (OVCA) cell lines, including TYKNU, M41-R, TYKNU-R, M41-R, PE01, A2780S, Skov3, OVCAR8, MCAS, A2780CP, HEYA8, and OVCAR5. In all cell lines, PPD treatment can significantly decrease cisplatin IC50.48 Paris polyphylla mixtures containing PPD inhibit the migration of LA795 cells in vitro and inhibit the tumor growth in vitro.37 PPVII induces death in different cell lines, including gastric cancer SNU-5, lung cancer A-549, skin cancer carcinoma A431, oral cancer OECM-1, breast MCF-7, pancreas MiaPaca-2, colon HTB-39, human normal fibroblasts (FR2).
Mechanistic Studies of PPs in Different Cancers
This section discusses those studies in which PP mechanisms are reported.
Apoptosis: PPs Induce Apoptosis in Different Cancers Through the Following Mechanisms
Oxidative stress is the disturbance in redox signaling and regulation or physiological imbalance in the production of reactive oxygen species (ROS), such as oxygen (O2) or hydrogen peroxide (H2O2), and the body’s ability to remove them.49 ROS are generated throughout the body as by-products of cellular aerobic metabolism, exposure to X-rays or ultraviolet light, and on-going stress.50 ROS play pivotal roles in cell signaling and the regulation of growth factors, transcription, cytokines, hormones, neuromodulation, apoptosis, and immunomodulation.50,51 ROS also function in different cell processes, including cell survival, proliferation, differentiation, gene expression, elimination of pathogens or foreign particles, and enzyme regulation.52,53 The high oxidative stress in cancer cells increases cell survival, proliferation, angiogenesis, and metastasis; disrupts cell death signaling; and causes drug resistance.54–56 Although ROS increase cell proliferation, they have been recently deemed useful in cancer treatment. Plant-derived compounds induce apoptosis in cancer cells by promoting ROS generation in these cells above the threshold level.54,56–58
Several PPs induce apoptosis in cancer cells through oxidative stress, which promotes the generation of ROS and the dissipation of mitochondrial membrane potential (MMP). In these PPs, PPI generates ROS and dissipates MMP in HCT 116 and MDA-MB-231 cells,59–61 PPII in HepG2,62 PPVI in HepaRG,63 PPG or PPVII in HepG-2 cells.64 Furthermore, PPD or PSI and PPG or PPVII cause dissipation of MMP in K562/A02 human leukemia drug-resistant and K562 cells65 and human NPC cells, respectively.66 ROS generation and MMP dissipation are reversed by the ROS inhibitor N-acetyl-l-cysteine (NAC) treatment.62–64 Oxidative stress is further summarized in Figure 1A.
The mitochondrial-dependent pathway is important for apoptosis induction, and any disturbance in this pathway prevents apoptosis. This pathway is regulated by B cell lymphoma-2 (Bcl-2) family proteins through changes in the permeability of the mitochondrial membrane for the release of different apoptotic proteins, including cytochrome-c (Cyt-c).67 Anti-apoptotic proteins, such as B cell lymphoma extra-large (BclxL), Bcl-2, Bcl-2-related protein A1, and myeloid cell leukemia 1, increase cell survival. Pro-apoptotic proteins, including Bcl-2 homologous killer, Bcl-2 associated X (BAX), and Bcl-2 homology domain 3 (BH3)-only proteins act as receptor mediators and cause apoptosis by inducing mitochondrial stress.68
BH3-only proteins have two subclasses: activators and depressors/sensitizers. Activators include total BH3 interacting domain death antagonist and Bcl-2 like protein-11. This subclass activates BAX/BAK directly and causes MMP depolarization. Depressor/sensitizers include Bcl-2-associated death promoter, Bcl-2-interacting killer, phorbol-12-myristate-13-acetate-induced protein-1, hara-kiri, and upregulated modulator of apoptosis p53. Instead of BAX/BAK activation, this subclass counterbalances anti-apoptotic proteins.69,70 Meanwhile, anti-apoptotic proteins block the death signaling pathway through the direct inhibition of activator BH3-only proteins or BAX/BAK activation.71 Anti-apoptotic proteins (Bcl-2 and Bcl-xl) cause cancer progression72 and therefore increase the resistance of tumor cells to different types of cell death stimuli, including anticancer drugs.70
In hepatocarcinoma cells, PPI and formosanin C synergistically downregulate BCL2 inhibition and BAX upregulation and release of cytochrome from the mitochondria. Cytochrome c release from the mitochondria activates caspase-3,9 and induces hepatocarcinoma cell apoptosis.73 In 143-B, MG-63, U-2 OS, non-small cell lung cancer (NSCLC), Saos-2, A549, SK-MES-1, H460, MDA-MB-231, MCF-7, U251, HepG-2, RPMI8226, SMC7721, and HO-8910M cells, PPI induces apoptosis through the mitochondrial-dependent pathway (MDP).74–83 In the MDP, PPI inhibits Bcl-274–79 and Bclxl75 while activating Bax74,75,78 and Bak,75 triggering Cyt-c release from the mitochondria to the cytosol.61,78,79 The PPI-induced release of Cyt-c in the cytosol activates caspase-9,61,82,83 caspase-3,74–77,80,82 and PARP.61,74–76,80,82 The activated PARP enters the nucleus and causes DNA damage, leading to cell apoptosis.77
PPII induces apoptosis in HepG2 cells through modulation of the mitochondrial pathway via downregulation of Bcl-2 and upregulation of Bax, triggering cytochrome c release from the mitochondria and caspase-9 activation. Activated caspase-9 further activates caspase-3 and PARP, respectively, resulting in apoptosis.62
In MCF-7 and MDA-MB-231 cells, PPD induces mitochondrial-dependent apoptosis through MMP dissipation,24 Bcl2 downregulation,24 and Bax upregulation,24 which further activate caspase-9,24 leading to cell apoptosis.24
In HepG2,25 R-HepG2,25 NB-69,84 K562/A02,65 U87 glioma,85 SGC790187, MCF-7,24 MDA-MB-231,24 and NSCLC cells, PPD or PSI induces apoptosis by modulating the mitochondrial-dependent pathway. In the mitochondrial-dependent pathway, PPD or PSI downregulates Bcl224,65,85–87 and upregulates Bax.24,65,85–87 As a result, the mitochondrial membrane becomes permeable, and cytochrome c65,86 and AIF25 are released from the mitochondria to the cytosol, causing the activation of caspase-9,84,86 caspase-3,25,65,84–87 and PARP24,25 and the apoptosis of cells.24,25 PPD-induced apoptosis was reversed by pretreatment with the inhibitor SP60012585 through modulation of Bcl-2,85 Bax,85 caspase-385 and with the caspase inhibitor z-DEVD-fmk through downregulation of caspase-3 in HepG2 and R-HepG2 cells.25
In H460,88 H446,88 and SGC-790189 cells, PSI sensitizes camptothecin-, 10-hydroxycamptothecin-, and cisplatin-89 induced apoptosis through modulation of the mitochondrial pathway. In the mitochondrial pathway, PSI inhibits Bcl288,89 and Bclxl88 while upregulating Bax,88,89 causing mitochondrial membrane permeability and cytochrome-C88 release from the mitochondria into the cytosol. As result, PSI increases caspase-988 and caspase-3 expression.88,89 Furthermore, PSI and hyperthermia at 43°C induce apoptosis in NSCLC cell lines through inhibition of Bcl2, Bax, and caspase-3 protein expression.90
PPVI in HepaRG cells induces MDP apoptosis through Bcl-2 downregulation and Bax upregulation, triggering cytochrome c release from the mitochondria and upregulating caspase-9,3 and PARP.63
PPVII66 or PPG64,91 induces apoptosis in SAS,91 OECM-192, HepG-2,64 and NPC66 cells through downregulation of Bclxl66,91 and Bcl264,66,91 and upregulation of Bak,91 Bad,64 Bax,64,66 Bim,91 and tBid91 levels. This phenomenon causes cytochrome c64 release from the mitochondria to the cytosol, which further increases the cleavage of caspase-364,66,91 and −9.64,66,91 z-VAD-FMK (a broad-spectrum caspase inhibitor) was used in the following experiments to clarify the relevance of PPG-induced cell death. PG combined with Z-VAD-FMK substantially increases the viability and decreases the apoptosis of HONE-1 and NPC-039 cells.66 Formosanin C and PPVII increase apoptosis in NCI‐H460 cells by downregulating Bcl-2 and upregulating Bax, caspase-3, −8, and 9.92 The summarized form of the mitochondrial-dependent pathway is depicted in Figure 1A.
Extrinsic Apoptotic Pathway
Tumor-necrosis-factor (TNF) family proteins, such as Fas or TNF receptor (TNFR-1), activate the extrinsic apoptotic pathway.93 Fas or TNFR-1 upregulates caspase-8 through Fas-associated death domain, generating a death signaling complex that activates caspase-3 and results in cell death.94,95 In different cancers, NPs regulate the extrinsic apoptotic pathway.96,97
PPII induces the apoptosis of HepG2 cells by modulating the extrinsic apoptotic pathway via upregulation of FAS, caspase-8, and caspase-3.62 PPVI induces apoptosis in a dose-dependent manner in HepaRG,63 A549,98 and NCI-H129998 cells by upregulating the expression of death receptor-3 (DR3),98 DR5,98 Fas,63,98 caspase-8,63 caspase-3,63,98 and cleaved PARP63,98 but downregulating the expression of decoy receptor-3 (DcR3).98 Pretreatment with Z-VAD-FMK (caspase inhibitor) on HepaRG cells increases the percentage of viable cells, indicating that PPVI induces cell apoptosis through the mitochondrial pathway and the Fas death-dependent pathway.63 PPVII or PPG induces apoptosis in a dose-dependent manner in human NPC,66 A549,98 NCI-H1299,98 HepG-2,64 SAS,91 and OECM-192 cells by upregulating the expression of DR3,98 DR5,98 Fas,98 caspase-8,64,66,91 cleaved PARP,98 and cleaved caspase-364,66,91,98 but downregulating the expression of DcR3.98 The summarized form of the extrinsic apoptotic pathway is depicted in Figure 1B.
Signal Transducer and Activator of Transcription-3 Pathway
Signal transducer and activator of transcription-3 (STAT3) regulates different cellular processes, including proliferation, differentiation, immune function, and survival.99 STAT3 is activated through its phosphorylation at tyrosine-705 (Y705) or serine-727 (S727).100,101 STAT3 can be activated by growth factor receptors, cytokine receptors, Janus activated kinases, sarcoma family kinases, and Abelson murine leukemia family kinases.102,103 STAT3 is expressed in different cancers 104,105 and leads to transformation, resistance to chemotherapy, and tumorigenesis.106 These findings show that STAT3 is a good therapeutic target in the development of new drugs against cancer.
In NSCLC cells, the overexpression of lncRNA-metastasis-associated lung adenocarcinoma transcript-1 (MALAT1) increases STAT3 expression, which causes resistance to gefitinib, whereas PPI reverses the MALAT1-induced overexpression of STAT3 and causes cell apoptosis.76 Another study demonstrated that PPI reverses the IL6/STAT3-induced resistance to erlotinib in HCC827 cells 108. PP7- or PPVII-induced apoptosis in HepG2 cells by downregulating STAT3.64 The STAT3 pathway is further summarized in Figure 1C.
Wnt/β-catenin pathway is an oncogenic pathway that plays a crucial role in cancer progression.108 β-catenin is an intracellular signal transducer that plays an important role in cadherin protein complex and activates the Wnt/β-catenin pathway during embryonic development and tumorigenesis.109–111 Following activation of this pathway, wnt binds to membrane proteins, including serpentine receptors, frizzled family, and low-density lipoprotein receptor-related protein-5/6, which is necessary for disheveled (Dsh/Dvl) phosphorylation. Activated Dsh/Dvl works at intermediate and transfer signals from the Wnt/β-catenin-receptor complex to axis and glycogen-synthase-kinase-3β (GSK-3β) to downregulate the phosphorylation of β-catenin.112–114 As a result of Wnt binding to its receptors, the unphosphorylated β-catenin accumulates in the cytoplasm. The accumulated β-catenin enters the nucleus, where its downstream target genes, such as c-myc, are activated.115 Furthermore, the Wnt-signaling pathway controls different cellular functions, including apoptosis, cell proliferation, migration, and invasion, which enhance the Wnt-dependent carcinogenesis.116,117
PPI inhibits cell growth, proliferation, and metastasis and induces apoptosis in 143-B, HOS, RPMI8226, and HO-8910M cells through the Wnt/β-catenin pathway. In the Wnt/β-catenin pathway, PPI inhibits Wnt5A and p-GSK-3β, further inhibiting β-catenin and its nuclear translocation.74,80,81,83 The PPI-reduced cell viability is abolished by GSK-3β specific inhibitor CHIR99021 while potentiated by β-catenin silencing.74 Furthermore, PPI inhibits the downstream regulator of b-catenin, such as surviving,80 as depicted in Figure 1D.
Cell cycle regulates cell growth at different checkpoints through the interaction of cyclin among exact cyclin-dependent kinases (CDKs), forming active complexes. The process ends before entering the new phase of the cell cycle.118 Then, CDKs are negatively regulated through different CDK inhibitors. CDK p21 controls the cell cycle at various checkpoints.119,120 Failed regulation of these checkpoints causes genomic rearrangement and mutation, resulting in genetic disturbance and ultimately cancer.120 Meanwhile, p53 is a key component that plays a crucial role in cell cycle regulation. A wide spectrum of damages and stresses activates p53.121,122 When p53 is activated through genotoxic stress, it controls the p21WAF1/CIP1/SDI1 genes encoding CDKs universal inhibitors, resulting in cell cycle inhibition.123 A plethora of research demonstrated that the anticancer compound causes death in cancer cells through induction of cell cycle arrest.124
In hepatocarcinoma, HCT 116, osteosarcoma 143-B, HOS, MG-63, U-2 OS, Saos-2, human myeloma RPMI8226, U266 cells, Du145, and PC3 cells, PPI causes cell cycle arrest in different phases, including G1, G2/M, S-phase, and G0/G1.59,73–75,78,80,125 In hepatocarcinoma cells, PPI and formosanin C synergistically upregulate p53, p21, and p27 and downregulate PCNA, CDK2, and Cyclin E, which lead to G1 phase cell cycle arrest.73 PPI in human myeloma RPMI8226 and U266 cells, U251 human glioma cells, HCT 116 cells 143-B and HOS cells induced ROS mediated G2/M phase cell cycle arrest through p21 upregulation which results in downregulations of cyclin B1, CD1 and c-myc.59,74,78,80 The ROS-mediated G2/M phase cell cycle arrest is reversed by NAC treatment.59 One study reported that PPI can cause S-phase cell cycle arrest in MG-63, U-2 OS, and Saos-2 osteosarcoma cells through downregulation of c-myc, Cyclin B1, Cyclin D1, and CDK1 time-dependently; this result suggests that the cell cycle arrest is due to cyclin/CD1 complex disturbance.75 Another study reported that PPI causes the G0/G1 phase cell cycle arrest in Du145 and PC3 cells with an unknown molecular mechanism.125
PPD or PSI induces G2/M phase cell cycle arrest in SGC7901,86 NSCLC,87 K562,65 and K562/A0265 through upregulation of CD1465 and p2165 and downregulation of cyclin B1,65,86 cyclin-dependent protein kinase,65 and Cdk1.86 PSI increases the cisplatin-induced G2/M phase cell cycle arrest in SGC-7901 cells through P21waf1/cip1 activation.89 PSI and hyperthermia at 43°C induce G2/M phase cell cycle arrest in NSCLC cell lines.90
PPVI induces apoptosis and G2/M phase cell cycle arrest in a dose-dependent manner in A549 and NCI-H1299 cells by upregulating the expression P53 and p21 Waf1/Cip1, which may inhibit cyclin B1 in NCI-H1299 cells.98 PPVI induces S phase cell cycle arrest in HepaRG cells by decreasing the expression of cyclin A2 and CDK2 and upregulating the expression of p21.63
In oral cancer OECM-1,91,126 SAS,91 NPC,66 A549,98 and NCI-H129998 cells, PPVII or PG induces cell cycle arrest at different stages, including G2/M,98,126 S-phase,91 and sub-G167 phase. PPVII induces G2/M phase cell cycle arrest by upregulating P53,98 p27,126 p21 Waf1/Cip1,98,126 Cdc251C,126 checkpoint kinases 1/2 (Chk1/2),126 and ataxia telangiectasia mutated126 and by downregulating cyclin B198,126 with no effect on p-Cdc21 (Tyr15).126 The G2/M phase cell cycle arrest due to p21and p27 has been confirmed through siRNA treatment.126 PPG or PPVII induces S-phase cell cycle arrest through the downregulation of Cyclin A and CDK2 expression in a dose-dependent manner. In addition, p21 and p27, inhibitors of the G1-to-S-promoting complex cyclin E-CDK2, significantly increase the protein level.91 However, the molecular mechanisms for sub-G1 phase cell cycle are unexplored.66 In gefitinib-sensitive PC-9 cells and acquired gefitinib-resistant H1975 cells, PPVII increases the sensitivity of gefitinib and induces G1 phase cell cycle arrest by upregulating p21 and downregulating CDK2, CDK4, Cyclin E, and Cyclin D1.127 The cell cycle is further summarized in Figure 2A–D.
Phosphatidyl-Inositol 3-Kinase/Protein Kinase-B/Mammalian Target of Rapamycin Signaling Pathway
The phosphatidyl-inositol 3-kinase/protein kinase-B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes cell survival and growth through different molecular mechanisms.128,129 This pathway is activated in different types of cancer through multiple mechanisms.130–133 For example, AKT becomes activated as the phosphorylation of two residues including serine 473 (Ser 473) and threonine (Thr 308) of AKT occurs.134 After activation, AKT enters the nucleus, where they change the activities of transcription regulating factor. PI3K/AKT signaling elevates mTOR expression, which is associated with poor prognosis. Different NPs cure cancer through inhibition of the PI3K/AKT/mTOR pathway.135
PPI inhibits the proliferation and metastasis in ovarian cancer HO-8910M cells by downregulating PI3Kc2b.83 PP II increases the sensitivity of drug-resistant PC-9/ZD cells to gefitinib through the PI3K/Akt/mTOR pathway, in which it inhibits PI3K, AKT, and mTOR, which further activate Bax, caspase-9, and caspase-3 and trigger cell apoptosis.136 The PI3K/AKT/mTOR pathway is further summarized in Figure 3A.
Endoplasmic Reticulum Stress
The endoplasmic reticulum (ER) plays a role in the synthesis, signaling, and sensing of eukaryotic cells. In performing these functions, the ER must regulate oxidizing and the Ca2+-rich folding environment. In the ER, Ca2+ buffering and protein folding are regulated by a number of chaperons, including calnexin, calreticulin, glucose-regulated protein GRP78 (BiP), and protein-disulfide isomerase. A number of pathophysiological conditions, including ER-Ca2+ depletion, viral infection, hypoglycemia, oxidative injury, and hypoxia, affect the ER homeostasis and cause ER stress. The ER responds to these path-physiological changes by activating integrated signal transduction pathways through unfolded protein response (UPR).137 The UPR controls the ER homeostasis by activating ER folding machinery components, controlling ER quality, and coordinating gene transcription and ER-associated degradation (ERAD) pathway. When ER stress increases, the UPR changes from pro-survival to pro-death response, which leads to the activation of intrinsic apoptosis.138 Mammals have various ER stress transducers, including activating transcription factor 6 (ATF6), inositol requiring enzyme 1, and protein kinase RNA like endoplasmic reticulum (p-ERK). The UPR has pro-survival and pro-apoptotic responses; pro-survival response activates ER chaperons, translation attenuation, and ERAD, whereas pro-apoptotic response activates C/EBP homologous protein-10 (CHOP)/GADD153 and caspase-12.139
PPI in MG-63, U-2 OS, and Saos-2 osteosarcoma cells dose-dependently activates the PERK branch of UPR, as determined by the increase in Bip expression, robust in eIF2α phosphorylation, and upregulation of ATF4 and GADD153 (CHOP) transcriptional factors.75
PPD induces the ER stress in human NSCLC NCI-H460 cell line by upregulating glucose-regulated protein 78 (Bip/GRP78), protein disulfide isomerase (PDI), and C/EBP homologous transcription factor (chop), triggering cell apoptosis.26 The ER pathway is further summarized in Figure 3B.
c-Jun N-Terminal Kinase Signaling Pathway
The c-Jun N-terminal kinase (JNK) pathway regulates various physiological processes, such as cell differentiation, death, survival, inflammation, protein expression, and proliferation. Any failure in this pathway causes a spectrum of diseases, including cardiac hyper-therapy, diabetes, cancer, asthma, and auto-immune diseases.140 JNK plays a major role in oncogenic changes. The JNK pathways eliminate apoptosis by downregulating Ras transformation.141 Different NPs induce apoptosis in cancer cells by regulating the JNK pathway.142–144
In human glioma U251 and ovarian cancer HO-8910M cells, PPI induces apoptosis through the JNK pathway by upregulating JNK and c-Jun.78,81,83 Furthermore, PPI inhibits tumor growth by upregulating c-Jun.81 PPD induces apoptosis in U87 glioma cells by upregulating NH2-terminal kinase (JNK) phosphorylation, which is reversed by JNK inhibitor SP600125; this result suggests that the apoptosis is due to JNK pathway regulation.85 The JNK pathway is further summarized and depicted in Figure 3C.
Nuclear Factor Kappa B Pathway
The nuclear factor kappa B (NF-kB) pathway is a complex pathway consisting of five homo- and hetero-dimers of the reticuloendotheliosis oncogene cellular homolog (Rel) family, such as RelA (p-65), RelB, c-Rel, NF-kB1 (p-50/p-65), and NF-kB2 (p50/p65).145 In cancers, the NF-kB pathway becomes dysregulated.146 Active NF-kB has been reported in different cancers, including breast, prostate, colon, liver, leukemia, lymphoma, and ovarian cancer.147–149 As the DNA becomes damaged, the NF-kB pathway becomes activated, which results in the activation of NF-kB targeted genes, including Cyclo-oxygenase-2 (COX-2)150 and inducible nitric oxide synthase (iNOS).151
These genes play pivotal roles in pro-survival anti-apoptosis. Therefore, NF-kB is a candidate for therapeutic resistance in different cancers. Different NPs have potential therapeutic efficacy against cancer by inhibiting NF-kB pathway activation in cancer cells.152
PPI alone or in combination with formosanin C or cisplatin inhibits cancer through the NF-kB pathway in different cancer cell lines, including hepatocarcinoma cells, osteosarcoma 143-B, and HOS cells.73,75,79 In the NF-kB pathway, PPI alone or in combination downregulates NF-kB p-6573 and IKBα. It also inhibits p65 translocation from the cytoplasm into the nucleus.73,75 Furthermore, this inhibition causes the downregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP-9), leading to cell inhibition.73,79 One study demonstrated that the PPI in Du145 and PC3 cells inhibits NF-kB p65 in the cytoplasm, which further inhibits MUCIN1 directly and through HOTAIR. Furthermore, PPI inhibits p65 in the nucleus and HOT transcript antisense RNA (HOTAIR), which inhibits MUCIN1 expression and decreases Du145 and PC3 cell proliferation.125
PPI inhibits the migration, invasion, and proliferation of PC3 and DU145 cells by inhibiting HOTAIR, which further inhibits DNA methyl-transferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2) expression. Inhibition of EHZ2 expression also inhibits cell proliferation.153 The NF-kB pathway is further summarized and illustrated in Figure 4A.
Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated-Kinase Pathways
The mitogen activated-protein kinase/extracellular signal-regulated kinase (MAPK/ERK) or Ras-Raf-MERK-ERK pathway possesses different cascades, but Ras-Raf-MERK-ERK 1 and 2 (ERK1/2) are mostly dysregulated in human cancer.154 This pathway regulates different cellular functions, including cell growth, differentiation, apoptosis, proliferation, migration, and senescence.155 The protein molecules of the MAPK/ERK pathway are activated through phosphorylation. Following activation, ERK enters the nucleus, where different transcription factors are activated. When these transcription factors are activated, they attach to the promoter region with different genes, including apoptosis inhibitory genes, cytokines, growth factors, and genes that increase cell proliferation.156 Dysregulation of this pathway causes drug resistance, tumorigenesis, and senescence,155,157,158 as detected in many human cancers.159,160 The MAPK pathway consists of ERK, JNK, and p-38MAK,161 which play a critical role in normal and cancerous cell proliferation and cause drug resistance.162,163 Small molecules targeting kinases in the MAPK pathway are not only useful in patients with NSCLC but also in patients with SCLC.164 NPs have a potential effect against cancer through MAPK pathways. Recently reported NPs, including PSI or PPD, PPVII or PPG have a potential antitumor effect through MAPK pathways.64,88,91
PSI or PPD sensitizes the CPT/HCPT-mediated inhibition of p38 MAPK and activation of phosphorylation of p38 MAPK in H1299 cells and the suppression of AKT and ERK pathway activation in H460 cells.88
PPVII or PPG induces apoptosis in HepG-2,64 OECM-192, and SAS91 cells by upregulating AKT,91 JNK,64,91 ERK,64,91 and p38.64,91 The involvement of the MAPK64,91 was evaluated using Akt inhibitor (LY294002),91 JNK inhibitor SP600125,64,91 ERK inhibitor PD98059,64,91 (5 μM), and p38 inhibitor SB20358064,91 to pre-treat HepG2,64 OECM-192, and SAS91 cells before the treatment of PP7. The MAPK64 (AKT, ERK1/2, p38 and JNK1/2)91 inhibitors could significantly reduce the expression of apoptosis-related proteins in HepG2, OECM-1,91 and SAS91 cells and decrease the apoptosis64,91 and necrosis64 of HepG2 cells treated with PP7.64
PPG-induced apoptosis in NPC cells through ERK1/2 and JNK1/2 and confirmed through the use of ERK1/2 inhibitor (U0126) increases the apoptosis and JNK1/2 inhibitor (SP600125), which inhibit the apoptosis caused by JNK upregulation.66 The PP anticancer effect through MAPK pathways is further summarized in Figure 4B.
AUTOPHAGY: PPs Induce Autophagy in Different Cancers Through the Following Mechanisms
PPs Induces Autophagy Through the PI3K/AKT/mTOR and AMPK/mTOR Pathways
A new target for cancer treatment is autophagy, which is activated through chemotherapy, nutrient deprivation, and oxidative stress and causes the degradation of damaged cytoplasmic organelles and proteins in response to external stress.165,166 Different signaling pathways are involved in autophagy, including adenosine mono-phosphate activated protein kinase (AMPK) and PI3K/AKT/mTOR pathways.167,168 The PI3K/AKT pathway serves as a positive regulator of the mTOR pathway and a negative regulator of autophagy in cancer cells169 , but the disruption of PI3K/AKT/mTOR pathways through anticancer agents results in autophagy. AMPK regulates energy homeostasis and causes autophagy through inhibition of mTOR complex 1.168,170 Furthermore, the JNK pathway is involved in the autophagy of cancer cells in response to pharmacological stress.171,172 A number of studies revealed that autophagy is triggered by inhibition of the PI3K/AKT/mTOR pathway through a link with activation of the JNK pathway.173,174 Autophagy protein Beclin-1 interacts with Bcl-2, forms Beclin-1/Bcl-2 complex, and inhibits autophagy.175 Activated JNK causes Bcl-2 phosphorylation, results in Bcl-2 degradation, dissociates Beclin-1 from the Beclin-1/Bcl-2 complex, and causes autophagy.176–179 Furthermore, JNK activation is essential for anticancer agent-induced autophagy cell death.176,180
One of the hallmarks of autophagy is the conversion of light chain 3I (LC3I) into its lapidated form LC3II, which is essential for autophagosome formation in autophagy.181 Another marker of autophagic flux is P62, which has an inverse relation to autophagy activity. It binds to LC3 and degrades in autophagy.182 Moreover, in the study of autophagy, a commonly used approach is the examination of autophagic flux and autophagosome formation. A critical crosstalk between apoptosis and autophagy is Bcl-2, which inhibits autophagy through binding with Beclin-1 that initiates autophagosome formation during autophagy.175
PPI induces protective autophagy through the PI3K/AKT/mTOR pathway by inhibiting mTOR (S2448), AKT (S473), 70-kDa ribosomal protein S6 kinase (p70S6K (T389)), and 4E (eIF4E)-binding protein 1(4EBP1 (T37/46)) and increasing LC3I conversion into LC3II. In SMMC7721 and HepG2 cells, EGF markedly increases the phosphorylation of Akt and p70S6K and reverses LC3II, which suggests that the autophagy is due to the PI3K/AKT/mTOR pathway.82
PPI induces ROS-mediated autophagy in HCT 116 cells by inhibiting mTOR (s2448) and AKT (S473), p62 expression and increasing LC3II expression.59 The increase in LC3II is reversed by NAC pre-treatment.59
PPVII or PPG treatment induces autophagy in HepG-2,183 HONE-1, and NPC-03966 cells via the PI3K/AKT/mTOR and AMPK/mTOR pathways. In the PI3K/AKT/mTOR pathway, PPVII inhibits the expression of PI3K,66,183 Akt183 and mTOR,66,183 activated mTOR (ser2448),66 total mTOR,66 Raptor,66 Rictor,66 and GβL66 in a dose-dependent manner.66,183 PPG-induced autophagic death is confirmed when Baf A1 and wortmannin (autophagy inhibitors) increase cell viability and PPG treatment alone exerts no effect on the apoptosis of HONE-1 and NPC-039 cells.66 PPVII also increases AMPK phosphorylation, which further inhibits mTOR and induces autophagy in HepG-2 cells through modulation of the AMPK/mTOR pathway.183 These mechanisms are further summarized in Figure 5.
PPs Induces Autophagy Through the Akt, p38MAPK, ERK1/2, and JNK Signaling Pathways
A new target for cancer treatment is autophagy, which is activated through chemotherapy, nutrient deprivation, and oxidative stress and causes the degradation of damaged cytoplasmic organelles and proteins in response to external stress.165,166 Previous research demonstrated that the AKT and MAPK pathways are involved in autophagy and apoptosis.172,184–186
PPG or PPVII induces autophagy through the Akt, p38MAPK, ERK1/2, and JNK pathways in HepG-2,183 NPC-039,66 OECM-1,91 and SAS91 cells. PPG or PPVII activates JNK1/266,91,183 and inhibits AKT,66 p38 MAPK,66 and ERK1/2,91 which further increases the conversion of LC3I to LC3II,66,183 P62 degradation,66,183 and formation of LC3-positive structures or LC3 puncta.66,183 Furthermore, PPVII treatment decreases total Bcl-2183 while increases p-Bcl-2183 and Beclin.66,183 The AKT inhibitor (LY294002)66 and JNK1/2 inhibitor (SP600125)66,183 decrease PPG-induced autophagy, whereas p38 MAPK inhibitor (SB203580)66 increases this process, suggesting that the PPG-induced autophagy is due to the Akt, p38 MAPK, and JNK1/2 pathways.66,183 Moreover, LY294002, U0126, and SP600125 significantly attenuate PG-induced LC3-II activation, suggesting that the activation of ERK1/2 and JNK1/2 is involved in PG-induced autophagy.91 These mechanisms of autophagy are further summarized in Figure 5.
Inflammation is a physiological response for the protection of the body from tissue injury or infection and plays a critical role in different types of human cancers and other chronic diseases.187 In innate immune system, macrophages are the immune cells that play a central role in inflammation and protect the body from harmful stimuli. Active macrophages play important roles in host defenses against pathogens. They exert phagocytic activities and enhance inflammatory responses by producing different inflammatory factors, such as nitric oxide (NO) and prostaglandin-E2; proinflammatory mediators, such as iNOS, nitrogen species, metalloproteinases, and COX-2; and pro-inflammatory cytokines, such as TNF-α, interleukin-6 (IL-6), and IL-1β, which trigger other immune cells to the infection site.188–190 The overexpression of inflammation-related cytokines and mediators by activated macrophages is associated with the pathophysiology of different inflammatory and autoimmune diseases.191–193 Lipopolysaccharide (LPS) is a potent macrophage activator and endotoxin derived from the cell wall of Gram-negative bacteria.194 When LPS stimulates the macrophages, the expression of cytokines and mediator is regulated by NF-kB through MAPKs, including JNKs, ERKs, and p38-MAPK.195,196 Macrophages secrete MMP-9, which controls leukocyte migration in inflammatory diseases.197 In macrophages, MMP-9 is regulated by LPS via the NF-kB and MAPK pathways.198–200 Therefore, targeting MAPKs, NF-kB, and MMP-9 through NPs may have a potential effect on the treatment of inflammatory diseases.
In RAW264.7 cells, PPVII inhibits the LPS-induced phosphorylation of p38, JNK, and ERK, which indicate the involvement of MAPKs in the suppression of cytokines and mediator by PPVII.199 Macrophage produces pro-inflammatory cytokines (major components of inflammation), including interleukin-6 (IL-6), IL-1β, and TNF-α, and the PPVII-inhibited productions of these cytokines are markedly increased in LPS-treated cells.199 Furthermore, PPVII inhibits IkB-α phosphorylation, increases the p65 level in the cytoplasm that is inhibited in the nucleus, and downregulates PGE-2, NO, iNOS, COX-2, and MMP-9 at the mRNA and protein levels in LPS-activated cells.199 The above discussion suggests that PPVII inhibits inflammation through the MAPK and NF-kB pathways, as illustrated in Figure 6A.
Cell death usually occurs through two main pathways, namely, programmed cell death or apoptosis200 and direct cell damage or necrosis. Programmed necrosis or necroptosis is a new type of necrosis that has been recently reported.201 Necroptosis mechanisms involve the initial formation of a complex that contains receptor-interacting protein kinase-1 (RIPK1) induced by LPS and TNF. Furthermore, a complex is formed among RIPK3, RIPK1, Fas bound death domain protein, and caspase-8.202 By contrast, when the caspase-8 is inactive, RIPK3 and RIPK1 become activated and a RIP3 substrate known as mixed lineage kinase domain-like (MLKL) is phosphorylated, which attach to the cell membrane and form a hole in the cell membrane that causes the destruction of the cell membrane via necroptosis.203–205
PPD-induced necroptosis in LA-N-2 and IMR-32 cells, which is confirmed when the cells are co-treated with necrosulfonamide (a specific inhibitor of RIPK3 associated with necroptosis), through inhibition of PPD-induced cell death,84 as illustrated in Figure 6B.
Mitochondrial autophagy or mitophagy is a cellular pathway that helps in the removal of damaged mitochondria.206,207 Mitophagy plays a crucial role in the control of cancer microenvironment, cancer cell survival, and death. The study of molecular mechanisms of mitophagy might be important for the development of new cancer therapies.208 The PIKK1/PINK2 pathway regulates mitophagy. PARK-2 is a RING domain-containing E3 ubiquitin ligase, which is activated via autoubiquitination.209 In depolarized state, mitochondria use the uncoupling reagents, including carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and PARK2 enters the mitochondria and promotes its degradation.210 Furthermore, PARK2 overexpression induces the degradation of depolarized mitochondria via mitophagy.211 Given that PARK2 also selectively binds only to damaged mitochondria, it might help ensure the specificity of mitophagy.212 PTEN-induced kinase 1 (PINK1) has a targeting sequence located in the mitochondria.213 PINK1 protects the mitochondria from neurotoxin-induced injury, whereas loss of PINK1 function or mutation causes ROS-mediated mitochondrial injury.214 The full-length expression of PIK1 increases CCCP-mediated mitophagy or autophagy.215 Under stress conditions, the depolarization of mitochondrial membranes prevents the mitochondrial uptake and processing of PINK1; therefore, the unprocessed PINK1 on the outer membrane of the mitochondria recruits PARK2 and eliminates the damaged mitochondria through mitophagy.210 Dynamin-related protein 1 (DRP1) inhibitor mdivi-1 prevents mitophagy by inhibiting the fusion–fission cycle, which shows the importance of mitochondrial fission in mitophagy.216 The mitochondrial fission mediated by DRP-1 causes LC3B lipidation and mitophagy, for which PINK1 and PARK2 are required.217 A recent study has demonstrated that the LC3B-II autophagosome targets the mitochondrial membrane through interaction with C18-ceramide-LC3B-II, increases lethal mitophagy, and inhibits tumor growth.218 The above description demonstrates that mitophagy might be helpful in the identification of new therapies for cancer treatment.
In MDA-MB-231 cells, PPI induces DRP1 translocation to the mitochondria through dephosphorylating of DRP1 at ser-637, causing mitochondrial fission. PPI increases the stabilization of full-length PINK1 at the surface of mitochondria, leading to P62, PARK2, ubiquitin, and LC3B-II recruitment to the mitochondria and mitophagy. PPI-induced mitophagy is suppressed markedly with PINK1 knockdown. DRP1 suppression through shRNA or mdivi-1 inhibits the knockdown of PINK1 and PPI-induced mitochondrial fragmentation. These results suggest that PINK1 depletion leads to fission and mitochondrial fragmentation,61 as shown in Figure 6C.
In vivo Anticancer Effect and Toxicity of PPs
PPI inhibits tumor growth in lung cancer tumor xenograft77 and xenograft orthotopic mouse model74 without any side effects.74 PPI inhibits ovarian tumor in nude mice by downregulating Wnt5a,81 in PC-9-ZD xenograft by inhibiting MALAT1 and STAT3 expression,76 and in prostate cancer xenograft mouse model by inhibiting HOTAIR, DNMT1, and EZH2 expression.153 Furthermore, in MDA-MB-231 xenografts, PPI inhibits tumor growth that is enhanced by PINK1 knockdown. These findings show that PPI might be a good therapy for cancer.61 PPI inhibits the vasculogenic mimicry and microvessel density in PLC and PLC/Twist1 cells. Furthermore, immunohistochemically staining showed that PPI inhibits the expression of Twist1, VE-cadherin, vimentin, VEGFR1, and VEGFR2 and increases E-cadherin expression in tumor xenograft.219 PPI also overcomes the erlotinib resistance in HCC827-tumor xenografts by inhibiting IL-6; decreasing mesenchymal markers FN1, VIM, and SNAIL; and upregulating epithelial marker E-cadherin 109. In the embryo of zebrafish, PPD decreases the formation of inter-segmental vessels.47 PPD markedly inhibits the tumor in MCF-7 bearing nude mice with no obvious toxicity to heart and liver tissue nor increases the level of plasma enzymes, including aspartate transaminase, alanine transaminase, lactase dehydrogenase, and creatine kinase.24 PPD-containing saponins inhibit tumor growth in H22 tumor-bearing mice.35 In A549 xenografts, PPVI inhibits the tumor by causing apoptosis and G/M phase cell cycle arrest through upregulation of DR3, DR5, and p53 and attenuation of cyclin B1.98 PPVII and gefitinib inhibit tumor growth in acquiring gefitinib-resistant xenograft compared with either treatment alone. Furthermore, in xenograft model, PPVII and gefitinib activate the expression of p21 while reducing the expression of CDK4, CDK2, Cyclin D1, and Cyclin E compared with either treatment alone.127 In the NPC orthotopic graft model, PPG inhibits tumor growth by downregulating Ki67.66
In vivo Anti-Inflammatory Effect and Toxicity of PPs
PP7 dose-dependently suppresses the formation of xylene-induced ear edema in mice, suggesting that PP7 can potently inhibit acute inflammation in vivo. PP7 inhibits cotton pellet-induced granuloma formation in a dose-dependent manner in mice, indicating that it also inhibits chronic inflammation.199
Zebrafish (Danio rerio) is a freshwater fish used as a vertebrate model organism for different inflammation studies because its acquired and innate immunity is highly similar to that of mammals. The transparency of zebrafish embryo and larvae allows the dynamic and noninvasive imaging of in vivo inflammation.220–224 In adult zebrafish, the most abundant leukocytes are neutrophils; in BACmpx::GFP transgenic zebrafish, larvae possess green fluorescence in neutrophils, allowing the observation of neutrophil distribution in zebrafish.225,226 The numbers of neutrophils circulating near the lateral line neuromasts allow the measurement of inflammation. Copper sulfate and Lip+o-polysaccharide induce inflammation in zebrafish.
PPVII strongly inhibits NO generation and decreases the heartbeat and the size of yolk sac edema in LPS-stimulated inflammation in zebrafish embryos.199 Furthermore, pre-treatment with PPVII reduces the recruitment of neutrophils to the injured area, suggesting the potential effect of PVII against inflammation.199
PPs, including PPI, II, III, VI, VII, and PPD, are derived from different plants. These PPs exhibit potential effects against various cancers through different mechanisms in vitro and in vivo. PPs show their therapeutic effect on different cancers through apoptosis, autophagy, necroptosis, mitophagy, and inflammation. Among PPs, PPI targets apoptosis and autophagy through the PI3K/AKT/mTOR pathway. Thus, PPI is a candidate therapeutic drug for cancers activated by this pathway. In addition, although PPs target different pathways, a specific link among all pathways is lacking. Thus, researchers need to focus and find the link among these pathways. These PPs are ideal therapeutic drugs, and further research on their anticancer effect is highly encouraged.
The study was financially supported by the National Natural Science Foundation of China (81373875) and Liaoning Provincial Program for Top Discipline of Basic Medical Science (2012CB518803).
All authors declare that they have no competing interests in this work.
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