Advances in research on the anticancer mechanism of the natural compound cucurbitacin from Cucurbitaceae plants:a review

Jing Liang,Dan Chen*

1Department of Pharmacology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin,China.

Background

The aggressiveness of various cancers and progressive tumor resistance and side effects of currently available chemotherapeutic drugs are the main concerns associated with cancer treatments used worldwide.Natural compounds extracted from traditional Chinese herbs(TCH)exert obvious anticancer effects against various cancers and thus are new potential anticancer agents.Increasing number of studies have shown that cucurbitacins,which are highly unsaturated tetracyclic triterpenoids produced mainly by plants belonging to Cucurbitaceae family[1],can be administered in combination with various chemotherapeutic drugs to reduce tumor resistance and enhance the efficacy of these drugs[2-4].Although most cucurbitacins have a similar four-ring scaffold structure,they show a diversity in substituents(Figure 1).Several types of cucurbitacins are available,of which cucurbitacins B and E are the main cucurbitacin types[5].Other cucurbitacin types are produced through enzymatic reactions under certain environmental conditions[6].

Cucurbitacins are present in some TCH such as Gualou(Fructus Trichosanthis),Tianhuafen(Radix Trichosanthis),and Tianguadi(Pedicellus Melo).An ancient book named Shennong Bencao Jing(1602 A.D.,Donghan Dynasty of China)has reported that TCH Tianguadi(Pedicellus Melo)and the dried fruit stalk of Cucumis melo L.have been used for treating jaundice because components present in these herbs induce vomiting and aid in expelling phlegm.In the early 1970s,cucurbitacins B and E were isolated from Tianguadi(Pedicellus Melo)[7].Some researchers have shown that cucurbitacins have hepatoprotective,anti-inflammatory, antidiabetic, and anticancer properties [8-10].Cucurbitacin capsule (approval number:Z20090820),a Chinese patented medication,are used for treating chronic hepatitis and primary hepatocellular carcinoma because of their ability to induce detoxification,reduce fever,and remove dampness and yellowing.

Recent studies have focused on the anticancer effects of cucurbitacins,including cucurbitacins B,E,D,and I,which are the most common cucurbitacin derivatives isolated from Cucurbitaceae plants.Many in vitro studies have shown that cucurbitacins inhibit cell proliferation,migration,and invasion and induce G2/M phase cell cycle arrest,autophagy,and apoptosis through different molecular mechanisms in various tumor cells[11,12].In addition,animal experiments have shown that cucurbitacins significantly inhibit tumor growth and metastasis in vivo[1,13-15],indicating that these compounds exert a wide range of anticancer effects.In this review,we have discussed the advances in research on the anticancer mechanisms of cucurbitacins.

Anticancer mechanisms of cucurbitacins

Cucurbitacins induce the apoptosis of cancer cells through various mechanisms

Apoptosis and cell proliferation are the fundamental mechanisms for maintaining a dynamic balance in a numberofcellsin thebody.Cancercellsare characterized by inhibition of apoptosis,which induces their unlimited proliferation.Therefore,induction of cancer cell apoptosis by using various treatments is an important strategy for anticancer therapy.Cucurbitacins induce apoptosis of various cancer cells through different pathways(Figure 2).The subsequent subsections discuss mechanisms through which cucurbitacins promote cancer cell apoptosis.

STAT3 signaling pathway.Signal transducer and activator of transcription 3(STAT3)is one of the most important member of the STAT family that mediates the JAK(Janus kinase)/STAT3 signaling pathway and is closely associated with tumor development and metastasis[16].STAT3 overexpression promotes cancer cell proliferation and migration and inhibits cancer cell apoptosis.Recent studies have shown that STAT3 is involved in the resistance of tumor cells to chemotherapeutic drugs [17-19].Severalstudies performed over the last few years have reported the effectofcucurbitacinson theSTAT3 signaling pathway,which is considered to be a selective inhibitor of the JAK/STAT3 pathway.

Liu et al.reported that cucurbitacin B reduces the phosphorylation of STAT3 and its downstream targets,including cyclin B1 and BCL-2(B-cell lymphoma 2),in human laryngeal Hep-2 cells[20].In breast cancer cells,cucurbitacin B inhibits STAT3 phosphorylation in a time-and dose-dependent manner.However,Kin et al.reported that cucurbitacin B inhibits ERK(Extracellular signal-regulated kinase) 1/2 phosphorylation before STAT3 phosphorylation in leukemia cells,indicating that STAT3 is not the only target of cucurbitacin B[21].Cucurbitacin I exerts antiproliferative effects by inhibiting STAT3 signaling in breast cancer,glioma,squamous cell carcinoma of the head and neck,and lung cancer cells[22,23].Moreover,cucurbitacin I inhibits tumor angiogenesis in breast cancer MDA-MB-468 cells by decreasing STAT3 phosphorylation[24].In lung cancer A549 cells,cucurbitacin I induces apoptosis through a JAK/STAT3-dependent pathway [25]. In mice inoculated with five human osteosarcoma cell lines,namely,143B,HOS,MG63,SAOS-2,and HUO9,cucurbitacin I inhibits tumor growth by inactivating STAT3,thus improving the survival of these mice[26].Michelle et al.suggested that cucurbitacin I is highly selective for the JAK/STAT3 pathway and does not inhibit other tumor survival pathways[22].However,another study reported that cucurbitacin I promotes the apoptosis of gastric cancer cells by inducing reactive oxygen species(ROS)production and not by targeting STAT3[14].Cucurbitacin D inhibits the nuclear translocation and transcriptional activity of STAT3 in breast cancer MCF7,SKBR3,and MDA-MB-231 cells[4,27].Cucurbitacin E induces the apoptosis of pancreatic cancer cells through the STAT3 signaling pathway [28]. Graness et al. reported that cucurbitacins exert anticancer effects not by increasing STAT3 activity but by increasing ROS and antioxidant levels,which promote cell death[29].

MAPK signaling pathway.MAPK(Mitogen-activated protein kinase)signaling pathway is an important pathway involved in cell proliferation,differentiation,and apoptosis.p38,ERK and JNK(c-Jun N-terminal kinases),are the important members of the MAPK family[30].Many studies have shown that various cucurbitacins induce apoptosis of cancer cells through the MAPK signaling pathway.Cucurbitacin B induces the apoptosis of osteosarcoma U-2 cells,lung cancer A549 cells,and human neuroblastoma SH-SY5Y cellsby inhibiting the activation of JNK, ERK1/2, and p38 in a dose-dependentmanner [31-33].Cucurbitacin E promotes the apoptosis of triple-negative breast cancer cells by increasing JNK activation and inhibiting ERK activation [34].Deng et al. found that low concentrations of cucurbitacin I induce cell cycle arrest in and apoptosis of gastric cancer cells by activating JNK,p38,and MAPK signaling and by increasing GSH/GSSG ratio and GADD45α expression,which forms a positive feedback loop and independently regulates p53 gene expression[14].However,a few studies have reported that cucurbitacin D inhibits the MAPK signaling pathway,which should be explored further.

NF-KB pathway.NF-KB (Nuclear factor kappa-light-chain-enhancer of activated B cells)family includes five proteins,namely,RelA,RelB,Rel,NF-KB1,and NF-KB2.NF-KB is a dimeric protein comprising p65 and p50 subunits,and sustained NF-KB activation promotes tumor cell proliferation and inflammation [35].Ku etal.found that cucurbitacin D increases the levels of NF-KB in the cytoplasm and inhibits the nuclear translocation of phosphorylated NF-KB to induce apoptosis.Cucurbitacin D also induces the apoptosis of doxorubicin-resistant breast cancer cells[4].Ding et al.found thatcucurbitacin D inducesapoptosisby inhibiting intracellular proteasome activity and by reducing NF-KB nuclear translocation and BCL-2 and BCL-XL expression[36].Treatment of glioblastoma cells with cucurbitacin I activates the NF-KB pathway by inducing the phosphorylation and nuclear translocation of the NF-KB p65 subunit,which occurs before the inhibition of STAT3[37].

Figure 1 Structures of various cucurbitacin derivatives

Figure 2:Signaling pathways involved in cucurbitacin-induced apoptosis

PI3K/AKT pathway.Many recent studies have shown that the activation of the PI3K/AKT signaling pathway plays an important role in many biochemical processes,including the proliferation and survival of breast cancer,cervical cancer,human osteosarcoma,and other cancer cells[38].Wang et al.found that cucurbitacin E inhibits the growth of human osteosarcoma cells both in vitro and in vivo through the PI3K/AKT/mTOR signaling pathway[39].Moreover,cucurbitacin E reduces phosphorylated AKT(Protein kinase B)and total AKT levels in triple-negative breast cancer cells[34].Cucurbitacin D also inhibits the PI3K/AKT signaling pathway in cervical and gastric cancer cells by decreasing PI3K and p-AKT(Ser473)levels[15,40].Cucurbitacin I induces the apoptosis of A549 cells by inhibiting the activation of ERK and phosphorylation of its downstream proteins mTOR(Mechanistic targetofrapamycin)and STAT3;however,it does not inhibit the PI3K/AKT pathway[25].

Other pathways.Some studies have reported that cucurbitacins promote cancer cell apoptosis through other mechanisms besides those mentioned above.A study showed that cucurbitacin B inhibits breast cancer growth both in vivo and in vitro by inhibiting Wnt and HER2/integrin signaling [41].Duangmano etal.suggested that cucurbitacin B promotes the apoptosis of human breast cancer cells by disrupting microtubule networks[42].Cucurbitacins also induce the apoptosis of human T cell leukemia Jurkat cells by disrupting cellular actin mechanics and by activating its key regulator cofilin[43].Cucurbitacin B increases the production of intracellular ROS in leukemia K562 cells,thus inducing their apoptosis[44].It also induces the apoptosis of colon cancer SW480 cells through a STAT3-independent but an ROS-dependent mechanism[45].Cucurbitacin E induces the apoptosis of cervicalcancer HeLa and CaSkicells by upregulating the expression of death receptor 5[46].Cucurbitacin I promotes the apoptosis of liver cancer HepG2 cells by activating p53 and its downstream targets. Cucurbitacin D effectively induces the apoptosis of gastric cancer cells by activating the inducible nitric oxide synthase pathway[40].These findings indicate that cucurbitacins use variable and complex mechanisms to induce the apoptosis of cancer cells.

Cucurbitacins induce autophagy in tumor cells through various mechanisms

Autophagy is an important process for maintaining homeostasis in eukaryotic cells.Autophagy signaling pathways are activated under certain circumstances to degrade damaged macromolecular substances and to provide energy for cell survival.In cancer therapy,autophagy plays a dual role,such as the inhibition of autophagy promotes cancercelldeath,whereas excessive autophagy leads to autophagic cell death[47,48].Studies involving various cancer cell lines have shown that cucurbitacins B,D,E,and I induce the production of ROS,which play an important role in mediating DNA damage and in inducing protective autophagy [44,49-54].Liu etal.reported that cucurbitacin B inhibits CIP2A/PP2A/mTORC1 signaling axis-induced autophagy in cisplatin-resistant human gastric cancer SGC7901/DDP cells[55].In vitro treatment of leukemia cells with cucurbitacin B induces autophagy as a survival response;however,specific mechanisms underlying this are unclear[43].Cucurbitacin E induces autophagy by downregulating the mTORC1 signaling pathway and by upregulating AMPK activity[56].Microtubule-associated protein light chain 3(LC3)is the key factor in autophagosome formation.Cucurbitacins D,E,and I induce autophagy by upregulating the LC3 gene expression in human gastric cancer cells,with the effect of cucurbitacin I being significantly higher than those of cucurbitacins D and E[57].Ni et al.found that cucurbitacin I induces damage-associated autophagy in gastric cancer A549 cells by inhibitingthe ERK/mTOR/STAT3 signaling pathway[25].

Competitive mechanisms of cucurbitacin-induced autophagy and apoptosis

Most studies on cucurbitacins have focused on their effect on promoting cancer cell apoptosis.The ability cucurbitacins to inhibit cell proliferation does not depend only on the STAT3 pathway.Many studies have reported that the morphological and biochemical features of cucurbitacin-induced apoptosis of human cancer cells are not apparent and that cell death is induced by ROS-mediated autophagy in most cucurbitacin-treated cancer cells.In recent years,cucurbitacin-induced autophagy has attracted considerable amount of attention; however,mechanisms underlying cucurbitacin-induced autophagy are not completely understood.Research has shown that cucurbitacin-induced autophagy competes with apoptotic signaling to limit the effect of apoptosis[58].Cucurbitacins induce cell-protective autophagy and other competing apoptotic mechanisms or increase apoptotic resistance.Some studies have shown that cucurbitacins induce autophagy based on damaged cellular morphology.Moreover,the damaged cellular morphology that induces pro-death autophagy differs from the damaged cellular morphology that induces apoptosis.We believe that cucurbitacins may act on signaling pathways that are common between apoptosis and autophagy but may show a different order of induction of both these processes.Furthermore,apoptosis and autophagy may activate or inhibitone anotheraftercucurbitacin treatment.Therefore,additional studies should be performed to determine the complex relationship between cucurbitacin-induced autophagy and apoptosis.

Cucurbitacins induce cytoskeletal destruction

Cucurbitacins induce morphological changes in cancer cells within a short period.Changes in actin filaments and microtubules play an important role in cancer cell proliferation,which is an important target of natural compounds used in cancer treatment[59].Wang et al.compared cucurbitacins B,E,and I with vincristine and colchicine and showed that the cucurbitacins interacted with the cytoskeleton and actin filaments to induce cell cycle arrest.In addition,cucurbitacins interfered with microtubule structure,thus affecting cellmitosis [60].Cucurbitacin E disrupts the cytoskeletal structure and inhibits the proliferation of prostate cancer cells[61].Cucurbitacin B covalently binds to cofilin,thus increasing actin depolymerization.However,some studies have shown that cucurbitacins do not directly bind to cofilin but inhibit the regulation of cofilin phosphorylation kinase to increase actin depolymerization and stimulate Rho/ROCK pathway to induce actin and phosphorylated myosin II co-aggregation [62,63].However,Zhang etal.suggested thatcucurbitacin B inducesthe actin aggregation through Gα13/RhoA/PKA/VASP signaling pathway[64].Although several studies have attempted to determine mechanisms underlying cucurbitacin-induced cytoskeletal destruction,these studies have provided different results.Therefore,specific mechanisms underlying cucurbitacin-induced cytoskeletal destruction are still unclear and should be determined by performing additional studies.

Cucurbitacins induce cell cycle arrest in cancer cells

A cell cycle involves a series of events in a cell from mitosis to the end of the next division.The length of a cell cycle reflects the state of a cell and is a cyclical process of cell material accumulation and cell division.Cancerous cells often show abnormal division cycles.Many studies have shown that cucurbitacins induce cell cycle arrest at different stages depending on the cell type.Cucurbitacin B arrests the cell cycle in the S phase in BEL-7402,HL60,and U937 cells and in the G2/M phase in Panc-1,MiaPaCa-2,K562,SW480,and Hep-2 cells[6].In pancreatic cancer cells,cucurbitacin B may induce cell cycle arrest in the G2/M phase by inhibiting JAK2,STAT3,and STAT5 activation;increasing p21 level;and decreasing cyclin A and cyclin B1 expression [65].In human hepatocellular carcinoma BEL-7402 cells,cucurbitacin B-induced cell cycle arrest in the S phase is associated with the inhibition of cyclin D1 and cyclin-dependent kinase-1 expression but is not associated with STAT3 phosphorylation[66].Most studies have suggested that cells arrested in the G2/M phase of the cell cycle are tetraploid cells that have undergone nuclear division but are unable to complete cytokinesis.Drugs that affectthe microfilamentskeleton induce various degrees of cell cycle arrest;therefore,cell cycle arrest induced by cucurbitacin B is most likely to be a consequence of damage to the microfilament skeleton[60].Multiple types of leukemia cells treated with cucurbitacin B show significant cell cycle arrest in the S phase[67].Kong et al.compared 12 natural drugs,including cucurbitacins B,E,and I,in four cancer cell lines and found that cucurbitacin E significantly reduced the viability of MAD-MB-468 and SW527 cells by regulating cyclin D1 and cyclin B expression,inhibiting phosphorylated STAT3 (p-STAT3),and activating p53 and p21,eventually leading to cell cycle arrest in the G2/M phase[34,68].Cucurbitacin D inhibits the proliferation of endometrial cancer and ovarian cancer cells and increased the ratio of these cells in the sub-G0/G1 and G2/M phases of the cell cycle[69].No FDA-approved drugs are available for treating neurofibromatosis type 2(NF2)-associated schwannomas and meningiomas.Spear et al.found thatcucurbitacin D exertsanticancereffectson NF2-deficient mouse schwannoma Sch10545 cells and human benignmeningiomaBen-Men-1 cellsby increasing the number of cells in the G2/M phase and inhibiting the proliferation of these cells,suggesting its potential as a therapeutic agent for treating these diseases[70].Jafargholizadeh etal.found that treatment of human gastric cancer AGS cells with cucurbitacins D,E,and I induces cell cycle arrest in the sub-G1 phase,eventually leading to cell death[57].These studies indicate that cucurbitacins play an important role in cell cycle arrest and that the effect of cucurbitacins on cell cycle arrest differs between different cell types.

Combinationtreatmentwithcucurbitacinsand chemotherapeutic drugs

The aggressiveness of various cancers and tumor resistance and side effects of currently available chemotherapeutic drugs are serious concerns associated with cancer treatment.Cucurbitacins,which are natural compounds,exert obvious antiproliferative effects on various tumor cells,indicating their potential asanticanceragents.STAT3 isassociated with resistance against anticancer drugs and is highly expressed in many drug-resistant cancer cells.Because cucurbitacins mainly target STAT3,several studies have examined the combination of cucurbitacins with various chemotherapeutic drugs and have shown that cucurbitacins reduce the resistance againstand enhance the efficacy of these drugs.The findings of these studies suggest that cucurbitacins are potential candidates for use in combination therapy with clinical anticancer drugs.

Cucurbitacins and chemotherapeutic drugs synergistically exert anticancer effects.Tang et al.found that cucurbitacin B synergistically increases the antitumor activity of doxorubicin by blocking the STAT3 pathway[2].Cucurbitacin B also enhances the anticancer effect of imatinib mesylate by inhibiting matrix metalloproteinase-2 expression in MCF7 and SW480 tumorcells [71].The combination of cucurbitacin B with gefitinib induces cell cycle arrest and apoptosis in human colorectal cancer cells through the EGFR and JAK/STAT pathways[3].Lee et al.showed that treatment with low doses of cucurbitacin B and methotrexate synergistically inhibit the AKT and mTOR signaling pathways in human osteosarcoma cells both in vivo and in vitro[72].El-Senduny et al.treated ovarian cancer A2780 cells and cisplatin-resistant A2780CP cells with a combination of cucurbitacin B and cisplatin or pretreated these cells with cucurbitacin B,followed by treatment with cisplatin.Results of contrast analysis performed in this study showed that the combination of cisplatin and cucurbitacin B decreased the levels of dual specificity tyrosine phosphorylation regulated kinase 1B,phosphorylated ERK1/2,and p-STAT3 and increased the level of ROS,thus significantly enhancing the effect of cisplatin on the ovarian cancer cells[73].Moreover, cucurbitacin B synergistically exerts antiproliferative effects along with cisplatin on cutaneous squamous cell carcinoma cells[74]and enhances the effectof arsenic trioxide-induced apoptosis by inhibiting STAT3 phosphorylation in lymphoma Ramos cells.In addition,the combination of cucurbitacin B and arsenic trioxide does not exert any proapoptotic effects on normal lymphocytes,indicating that this combination is non-toxic against normal blood cells.Experiments involving an in vivo nude mouse lymphoma model have further confirmed this synergistic effect of cucurbitacin B and arsenic trioxide[75].Ku et al.found that cucurbitacin D promotes the apoptosis of doxorubicin-resistant breast cancer cells through STAT3 and NF-KB[4].Chang et al.found that cucurbitacin I increases the sensitivity of medulloblastoma-derived cancer stem cells to apoptosis induced by chemotherapeutic drugs targeting STAT3[76].

Cucurbitacins can be safely used in combination with chemotherapeutic drugs.Myelosuppression and hepatorenal toxicity are the common side effects of chemotherapy.Ahmed etal.showed thatthe combination of cucurbitacin B with chemotherapeutic drugs did not increase toxicity in immunosuppressed mice undergoing orthotopic transplantation of breast cancer.Neurosensory and neuromotor toxicities are common in patients treated with docetaxel and other taxanes.However,it was found that treatment with the combination of docetaxel with cucurbitacin B was associated with lower neurotoxicity than treatment with docetaxel alone.These findings suggest that cucurbitacins exert protective effects against docetaxel-induced neurotoxicity;however,specific mechanisms underlying this effect of cucurbitacins are unknown[77].Previous studies have reported that cucurbitacin E can be used in combination with doxorubicin for treating ovarian cancer and enhances the efficacy of doxorubicin.An in vitro study has shown that cucurbitacin E increases doxorubicin level in M5076 ovarian sarcoma cells by suppressing doxorubicin efflux[78].Sadzuka et al.found that cucurbitacin E can be used in combination with doxorubicin for treating ovarian cancer to enhance the efficacy of doxorubicin without increasing its side effects.Asmentioned previously,cucurbitacin E inhibits the efflux of doxorubicin from the M5076 ovarian sarcoma cells,thus significantly increasing its concentration in tumor cells and reducing its concentration in normal cells.Tumor cells and normal cells show differences in membrane transporter expression;thus,the differencesin therole of cucurbitacin E between tumor and normal tissues may be becauseofdifferencesin theexpression of multidrug resistance-associated proteins between these tissues[36].

Conclusion

Cucurbitacins are natural compounds with various pharmacological activities,and their antitumor effects have received increasing attention.These compounds can prevent the proliferation of different tumor cells by inducing apoptosis,cell cycle arrest,autophagy,and cytoskeletal disruption (Table 1).Although many studieshave reported the antitumoractivity of cucurbitacins,mechanisms underlying this activity of cucurbitacins are unclear. Cucurbitacins exert anticancer effects both in vivo and in vitro through multiple targets,indicating their enormous anticancer potential.Thecombination ofcucurbitacinswith chemotherapeutic drugs also exerts strong synergistic anticancer effects.However,determination of the antitumoreffectsofcucurbitacinsischallenging because of the complex mechanism of tumorigenesis.Therefore,additional in-depth studies on cucurbitacins should be performed to confirm their potential as drug candidates for cancer treatment in the clinical setting.

Table1:Cucurbitacins inhibits proliferation of different cell lines

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