Induction of Apoptosis:
Apoptosis is programed cell death that plays important role in the maintenance of body homeostasis. Abnormal apoptosis leads to the development and progression of many diseases, including neoplasms and autoimmune diseases. Cucurbitacins promote apoptosis through multiple molecular mechanisms. Cucurbitacin B, D, E, I, and IIa induce apoptosis in various types of cancer cells by inhibiting the STAT3 pathway. STAT3 is a transcription factor that controls gene expression through cross-talk with other transcription factors, such as β-catenin, hypoxia-inducible factor-1α (HIF-1α), nuclear factor-κB (NF-κB), c-myc, c-jun, etc and blocking STAT3 activation induces apoptosis (Gorski, Jaworski, Shannon, & Robinson, 1985). Cucurbitacin B have marked morphological changes as a marker for apoptosis, which include nuclear fragmentation, chromatin condensation, and formation of apoptotic bodies. However, it seems that the inhibition of STAT3 by cucurbitacin B is cell type-dependent and the antiproliferative effect of cucurbitacins is independent of the STAT3 pathway. Cucurbitacin B may be useful for both modulating the sensitivity of cancer cells to cytotoxic lymphocyte and promoting anticancer immunity through the pharmacological inhibition of the JAK2/STAT3 pathway (Oleszek, 2002). By suppressing the JAK/STAT pathway and decreasing Bcl-XL expression. Cucurbitacin B induces apoptosis in pancreatic cancer cells. These mechanisms indicate that inhibition of the JAK/STAT3 pathway by cucurbitacin B may be effective in cancer immunotherapy (Rice, Rymal, Chambliss, & Johnson, 1981). Moreover, in the cells of human colon adenocarcinoma, the cucurbitacin B-induced apoptosis is controlled by a reactive oxygen species (ROS)-dependent, but a STAT3-independent mechanism. Cucurbitacin B suppresses cancer progression by the inhibition of tumor necrosis factor (TNF)-induced expression of NF-κB-dependent anti-apoptotic proteins and transactivation activity of RelA/p65, which include TRAF1, TRAF2, c-IAP1, and c-IAP2. Furthermore, cucurbitacin B inhibits phosphorylation of ATP citrate lyase (ACLY), an important enzyme for cancer metabolism, leading to poly ADP ribose polymerase (PARP) cleavage, accumulation of sub-G0/G1 cell population and activation of caspase 3 and 7 (Gorski, Jaworski, Shannon, & Robinson, 1986). Cucurbitacin B also inhibits HER2 and integrin signaling in breast cancer. In addition, in breast cancer cells, cucurbitacin B promotes cell cycle arrest in G2/M phase and apoptosis by down-regulating Wnt-associated signaling molecules, including β-catenin, cyclin D1, galectin-3, c-Myc, and phosphorylated GSK-3β. Similarly, cucurbitacin E inhibits cell proliferation by suppressing Wnt/β-catenin signaling by up-regulating the cancer suppressor gene Menin. Cucurbitacin D activates the apoptotic pathway by suppressing STAT3 activity in breast cancer cells and cleaving fragments of procaspase-3 and -9 and PARP in human endometrial and ovarian cancer cells (Rubinfeld et al., 1993). Moreover, cucurbitacin D is considered as a potential anticancer drug treated on neurofibromatosis type 2 (Nf2)-deficient schwannoma as well as meningioma because of its proapoptotic effects through inhibiting expression of phospho-PRAS40 and phospho-Akt. Cucurbitacin IIa is a class of anticancer drug with decreasing survival in cancer cells by deranging actin cytoskeleton and mediating cells to go through PARP-mediated apoptosis via the suppression of survivin, which is a downstream protein in the JAK2/STAT3 pathway (Metcalf, Metcalf, & Rhodes, 1980). In pancreatic and ovarian cancer cells as well as squamous cell carcinoma, cucurbitacin E promotes the cleavage of caspase-3 and inhibits the phosphorylation of STAT3, but not extracellular signal-regulated kinase (ERK)-1/2. Apoptosis triggered by cucurbitacin E is accompanied by up regulating truncated BID and Fas/CD95 as well as down-regulation of Delta Psi (m), resulting in releases of apoptotic protease activating factor 1, cytochrome c and apoptosis inducing factor and serial activation of caspase-9, -8 as well as -3 (Lavie & Glotter, 1971). Cucurbitacin I, a JAK/STAT3 inhibitor, enhances apoptosis by inducing cleavages of PARP and caspase-3, -7, -8, and -9 in colon cancer. Cucurbitacin I also effectively suppresses the STAT3 pathway and expression of Bcl-2 and survivin in head and neck squamous cell carcinoma cells. With the role of a tyrosine kinase inhibitor, cucurbitacin I regulates proteasome degradation (Halaweish & Tallamy, 1993).

Induction of Autophagy:
Cucurbitacins, especially cucurbitacin B and I, trigger autophagosome formation as well as the accumulation and conversion from light chain 3-I to LC3II in many cell types mainly through increasing production of mitochondrial-derived ROS and subsequently activating ERK and JNK. Activation of the AMP-activated protein kinase/ mammalian target of the p70S6K pathway, but not the PI3K/Akt pathway, occurs in cucurbitacin I-induced autophagy in glioblastoma. Glioblastoma treated with cucurbitacins and also has a decreased level of HIF-1α (Bauer & Wagner, 1983)

Induction of Cell Cycle Arrest:
In human cells, cell cycle transition is controlled by holoenzymes consisting of both regulatory (cyclin) and catalytic (cyclin-dependent kinase (CDK)) subunits. CDK inhibitors CDK1, p21=Waf1 and p27KIP1 act as key regulators of the cell cycle by binding to CDK complexes and decreasing kinase activity. Cucurbitacins induce cell cycle arrest by modulating multiple signaling pathways (Bowman et al., 1999). Cucurbitacin B induces G2/M cell cycle arrest in various cancers, including osteosarcoma (OS) cell, non-small cell lung cancer, breast cancer, cutaneous squamous cell carcinoma, glioblastoma multiform, laryngeal squamous cell carcinoma and pancreatic cell (Turkson & Jove, 2000). In hepatocellular carcinoma cells, the Cucurbitacin B-induced cell cycle arrest in S phase is related to a significant decline in cyclin D1 and CDC2 levels, and no change in cyclin B1 level, which is different from the other cell lines, indicating that cucurbitacin B is probably cell specific in mediating inhibition of cell growth. In breast cancer cells, Cucurbitacin B arrests cell cycle by inhibiting telomerase via the down-regulation of human telomerase reverse transcriptase and c-Myc, as well as up-regulation of p21, Waf1 and p27Kip1 expression via the defective gene BRCA1. In addition, Cucurbitacin B down-regulates protein expression of cyclin B1 and CDC25C without reducing CDK1 expression in colon adenocarcinoma SW480 cells nor increasing p53 and p21 expression in neuroblastoma .In non-small lung cancer cells, Cucurbitacin B exerts its effect on cell cycle arrest through the down-regulation of CDK1, cyclin D1, and PCNA proteins. Cucurbitacin I decreases the expression of cell cycle proteins including cyclin B1, cyclin A, CDK1, and CDC25C in colon cancer cells (SW480). These results indicate that different cell types treated with cucurbitacins may not use the same mechanism to trigger cell cycle arrest (Bowman, Garcia, Turkson, & Jove, 2000). Furthermore, the microtubule cytoskeletons are relevant to cell motility and cytokinesis in various cancers.

The formation of cofilin-actin rods induced by cucurbitacin B is regulated by chronophin in dependent, but cofilin phosphatase Slingshot homolog 1-dependent hyper activation of cofilin in melanoma, leading to cancer cell growth arrest and inhibition of migration also, cucurbitacin B significantly changes cytoskeletal network in breast cancer and glioblastoma multiforme cells, which induces incorrect polymerization of microtubule network and fast morphologic changes (Dong et al., 2010). In the cells treated with cucurbitacin B, the G-actin pool is rapidly depleted and actin aggregates are quickly formed through the activation of a ROS dependent mechanism that regulates various cytoskeleton-regulatory proteins, ultimately leading to anticancer effects. Similar to cucurbitacin B, cucurbitacin IIa is a class of anticancer agent that inhibits cell survival in cancer by embroiling the actin cytoskeleton. Cell cycle arrest in G2/M phase was induced by cucurbitacin D and IIa in breast cancer. In Nf2-deficient meningioma and schwannoma cells, cucurbitacin D decreases cyclin A, B, and E and inhibits expression of phospho-PRAS40 and phosphoAkt (Duncan, Duncan, Alley, & Sausville, 1996). Furthermore, cucurbitacin D increases p21Waf1 as well as p27Kip1 proteins and decreases cyclin A and B in endometrial and ovarian cancer cells. In human bladder cancer T24, pancreatic, ovarian, and breast cancer cells, cell cycle arrest in G2/M phase induced by cucurbitacin E is related to a significant increase in the levels of p27, p21, and p53 along with inactivation of STAT3, CDK1, and cyclin B. Additionally, cucurbitacin E blocks cyclin B1/CDC2 complex formation and induces the expression of GADD45 in colorectal and human brain malignant gliomas cells, indicating that the delay of mitosis by cucurbitacin E is regulated by the overexpression of the GADD45 gene family (Duangmano et al., 2010).

Inhibition of Cancer Invasion and Migration:
Cancer metastasis is a complicated process including cell migration, adhesion, and proteolysis of the basement membrane (BM) and extracellular matrix (ECM). In particular, the matrix metalloproteinase (MMP) family accounts for degradation of the compounds in BM and ECM. Cucurbitacin B markedly suppresses cell migration and invasion induced by 12O-tetradecanoylphorbol 13-acetate by inhibiting the phosphorylation of Akt, p38, and ERK1/2, and the down-regulation of MMP-9. Cucurbitacin E suppresses breast cancer metastasis by disrupting Arp/23-dependent actin polymerization and blocking the Src/FAK/Rac/JNK/MMP signaling pathway. In nasopharyngeal carcinoma (NPC), Cucurbitacin I reduces the invasiveness of NPC cells and possesses potent anoikis-sensitization activity against NPC. Additionally, cucurbitacin I suppresses the activation of Rac1 in breast cancer cells through a Jak2independent and ROS-mediated mechanism that alters the balance between Rho and Rac, which are important factors for the metastatic dissemination and transformation of cancer cells (Jayaprakasam, Seeram, & Nair, 2003). Cucurbitacins have been reported mainly in the Cucurbitaceae, are a class of highly oxidized tetracyclic triterpenoids possess the biogenetically unusual 10α-cucurbit-5-ene [19(10 →19β) abeo-10α lanostane skeleton which are well known for their cytotoxic activity (Escandell et al., 2007). The ethanolic extract of air-dried fruits of C. pepo was subjected to chromatography to give cucurbita glycosides A and B. In vitro assay both compounds showed weak cytotoxic activity against HeLa cells with IC50 values of 17.2 and 28.5 µg/ml respectively (Park et al., 2004). Treatment with cucurbitacins B and E showed growth inhibition accompanied by apoptosis and cell cycle arrest in breast cancer cell lines (MDAMB-231 and MCF-7). They also modulated the expression of proteins involved in cell-cycle regulation in both of the estrogen-independent (MDA- MB-231) and estrogen-dependent (MCF-7) in human breast cancer cell lines (Yuan, Wahlqvist, He, Yang, & Li, 2006)

Mechanisms of MAPK pathway for signaling network

Fig. 5: Mechanisms of MAPK pathway for signaling network (Zhang & Liu, 2002)

Growth inhibition and cytotoxic effect of cucurbitacin B on breast cancer cell lines SKBR-3 and MCF-7 were attributed to G2/M phase arrest and apoptosis. Cucurbitacin B treatment inhibited Cyclin D1, c-Myc, and β-catenin expression levels, translocation to the nucleus of β-catenin and galectin-3. Western blot analysis showed increased PARP cleavage suggesting induced caspase activity and decreased mutagenic Wnt-associated signaling molecules galectin-3, β-catenin, c-Myc, and cyclin D1 with changes in phosphorylated GSK-3β levels (Esterbauer, 1993)[39]. (Cu B) and cucurbitacin E (Cu E) inhibit Wnt and STAT3 signaling pathways; Cu B inhibits HER2 and integrin signaling pathways and elevates intracellular level of ROS; cucurbitacin D (Cu D) inhibits STAT3 activation; and cucurbitacin IIa (Cu IIa) inhibits survivin. To trigger autophagy, cucurbitacin I (Cu I) increases intracellular level of ROS. To inhibit cell migration and invasion, Cu E and Cu I inhibit Rac1 activation, and Cu B inhibits phosphorylation of ERK1/2, p38, and Akt. To induce cell cycle arrest, Cu B, Cu D, and Cu E down-regulate protein expression of key regulators of cell cycle, including cyclin B1, etc.

Table. 2: Compounds structure and their effectiveness on cancer cell lines

 Compounds structure and their effectiveness on cancer cell lines

In the past cucurbitacins have been viewed as toxic compounds with potential cytotoxic and anti-ecdysone effects. More recently, different researchers have established the selective role of some cucurbitacins as inhibitors of specific transcription factors. This new understanding of the activity and new data on the cytotoxicity of different cucurbitacins has led to the discovery of several interesting structural relationships which help avoid negative side effects, such as the presence of an acetoxyl group at C25, the double bond at C23, or the presence of a carbonyl or hydroxyl at C3, which can modify both the cytotoxicity and the pharmacological activity of cucurbitacins. The most interesting structures for obtaining new cytotoxic compounds are probably cucurbitacin B and E whereas the deacetyl-derivatives (e.g. cucurbitacin R) show the highest potential as the basis for a series of anti-inflammatory cucurbitacins.  The different studies conducted on cucurbitacins and the similarity of the results shows that these compounds are a selective kind of JAK/STAT pathway inhibitor and that they may be highly interesting for treating cancers in which this pathway is implicated, such as human lung adenocarcinoma, glioblastoma multiform, nasopharyngeal carcinoma, or chronic lymphocytic leukemia Many studies have postulated that the concomitant use of different cucurbitacins, especially I and B, could improve the effects of gemcitabine, methotrexate, docetaxel, or cisplatin, which could allow for a possible reduction in dosage in order to avoid the high number of undesirable effects of these drugs. For these reasons, pumpkin seed show promise as anticancer agents, especially in combination with other therapeutic agents when chemo-resistance and radio-resistance constitute a serious problem for the patient. Cucurbitacins thus have a great potential for use as pharmacological agents in the prevention and treatment of cancer. In addition, they constitute a selective laboratory tool for studying compounds implicated in the JAK/STAT pathway and they will also be beneficial for database development and template design for future drug development. The purpose of this review is to gather the information about an anticancer potential related to these highly diverse group of Cucurbitacin which may be useful in future research.

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