Cucurbitacins as Potent Chemo-Preventive Agents: Mechanistic Insight and Recent Trends

Cucurbitacins constitute a group of cucumber-derived dietary lipids, highly oxidized tetracyclic triterpenoids, with potential medical uses. These compounds are known to interact with a variety of recognized cellular targets to impede the growth of cancer cells. Accumulating evidence has suggested that inhibition of tumor cell growth via induction of apoptosis, cell-cycle arrest, anti-metastasis and anti-angiogenesis are major promising chemo-preventive actions of cucurbitacins. Cucurbitacins may be a potential choice for investigations of synergism with other drugs to reverse cancer cells’ treatment resistance. The detailed molecular mechanisms underlying these effects include interactions between cucurbitacins and numerous cellular targets (Bcl-2/Bax, caspases, STAT3, cyclins, NF-κB, COX-2, MMP-9, VEGF/R, etc.) as well as control of a variety of intracellular signal transduction pathways. The current study is focused on the efforts undertaken to find possible molecular targets for cucurbitacins in suppressing diverse malignant processes. The review is distinctive since it presents all potential molecular targets of cucurbitacins in cancer on one common podium.


Introduction
Cancer is a huge global threat, representing a heavy burden to social systems and health care sectors all over the world. Moreover, over the past decades, both the incidence as well as mortality rates of malignant disorders have been increased, with a continuous sharp rise expected for further years [1]. This situation clearly indicates that current therapeutic tools for combating cancer are insufficient, inducing often also a wide range of adverse effects to the patients with already weakened health status [2][3][4]. Therefore, novel, safe and more efficient treatment modalities are highly needed and must be developed, whereas one possibility for this is to focus more on the natural plant-derived agents [5].
In ethnomedicine, natural products have been used for the management of both benign as well as malignant neoplasms already for centuries [6][7][8][9]. This approach has led to Biomolecules 2023, 13, 57 2 of 18 launching the screening program of plant-derived compounds in the US National Cancer Institute in the middle of the 20th century, resulting in the isolation and development of several anticancer drugs such as vincristine, vinblastine, and paclitaxel among others, currently approved for clinical use against different cancer types [10][11][12][13]. This success clearly demonstrates that nature is an important resource for anticancer agents, encouraging to continue such efforts.
Cucurbitacins (designated by the letters A, B, C, D, E, F, G, H, I, J, K, L, O, P, Q, R, S) constitute a class of natural triterpenoids [14]. These compounds can be found in many plants from the genera Bryonia, Cucurbita, Cucumis, Echinocystis, Luffa, Citrullus and Lagenaria, conferring a bitter taste in cucumber [14,15]. Several recent preclinical studies have demonstrated that this group of phytochemicals can exert antitumor activities in a variety of experimental models of different malignancies, including lung cancer [16], gastric cancer [17], colorectal cancer [18], liver cancer [19], pancreatic cancer [20], ovarian cancer [21], cervical cancer [22] and melanoma [23]. Such anticancer effects are achieved via interaction of cucurbitacins with multiple molecular targets and intervening in diverse cellular signaling cascades, suggesting that a high potential of this compound should be developed as a therapeutic tool.
To appreciate and draw more attention to these ancient molecules, this review article is focused on the different anticancer activities of cucurbitacins in diverse experimental model systems, describing anti-inflammatory, cell-cycle arresting, proapoptotic, antiangiogenic and antimetastatic effects of these triterpenoids. Moreover, co-effects of cucurbitacins with conventional cancer drugs are considered, presenting the most potent combinations for further studies. In addition, the possibilities to overcome the low bioavailability issues characteristic for natural compounds by modern nanotechnological methods are also discussed. In this way, the present review provides a strong basis for moving on with in vivo animal studies and human clinical trials, hopefully to apply cucurbitacins in the clinical settings in the future.

Absorption and Metabolism of Cucurbitacins
The presence of cucurbitacins is observed mainly in plant roots and fruits. They are a highly diverse group and divided into 12 categories, cucurbitacins A-T [25]. Less research has been conducted on cucurbitacins' absorption, distribution, metabolism, and excretion; this is a topic that should be investigated considering the compound's potential toxicity to mammals. [14]. Oral administration of cucurbitacin B (CuB) has been studied and shown to be slowly absorbed and metabolized in the gut [26]. CuE at a concentration of 100-200 µg/kg treatment had a plasma half-life of about 58-72% [27]. Similarly, oral bioavailability of CuB has been studied to be~10% with plasma concentration ranging from 4.85 to 7.81 µg/L after 30 mins of oral dosing. After intravenous administration, it is distributed in large volume~51.65 l/kg and exhibits a high tissue to plasma concentration ratios of~60-280-folds in many organs. A negligible amount (~1%) of CuB was detected in urine and feces, and it was suggested that it probably undergoes biotransformation prior to excretion [28]. Studies have shown that they reach highest plasma concentration within 1.75 h and an elimination half-life of~2.5 h. As oral delivery and absorption of cucurbitacins remain a great challenge, recent focus has been on the use of nano-micelles co-modified with cucurbitacins, which enhances the relative bioavailability of CuB by Biomolecules 2023, 13, 57 4 of 18 2.14-3.43 times [29]. However, further pharmacokinetic studies comprising metabolism and distribution of cucurbitacins are still required.

Apoptotic and Cell-Cycle Arrest
Apoptotic cell death can be triggered in cancer through internal and extrinsic processes, which converge on the control of caspase-dependent proteolysis of cellular proteins and DNA fragmentation [30][31][32]. Similarly, all tumor types have abnormal cell-cycle progression activity, which acts as a catalyst for carcinogenesis [33]. Recent research has shown that a variety of biological processes are regulated by cell-cycle proteins [34][35][36]. Therefore, numerous chemo-preventive FDA-drugs have been shown to mediate antitumor effects either via activation of apoptotic or cell-cycle arrest ( Figure 2) signaling pathways [37][38][39]. For instance, results from  revealed that cucurbitacin I caused lung cancer (A549) cells to undergo excessive ERS, CHOP-Bax and caspase-12-dependent ERS-associated apoptosis [40]. In colorectal cancer (SW480 and Caco-2) cells, treatment with cucurbitacin B resulted in cell-cycle arrest at the G1 phase as well as decreased Cyclin D1 and Cyclin E1 levels. Both CRC cell lines underwent in vitro cell death when exposed to CuB, which was accompanied by caspase-3 and cleaved PARP [41]. Using triple negative breast cancer (TNBC), cucurbitacin E strongly boosted JNK activation while considerably decreasing AKT and ERK activation in MDA-MB-468 cells. It also significantly decreased expression of Cyclin D1, Survivin, XIAP, Bcl2 and Mcl-1 [42]. In the pancreatic cancer cell line Capan-1, CuD induced cell-cycle arrest and death via the ROS/p38 pathway [43]. Cucurbitacin I-induced cell death in ovarian cancer (SKOV3) included apoptosis, as evidenced by upregulated caspase 3 and BAX and a decrease in Bcl2 [21]. Flow cytometric measurement of DNA content and RT-PCR analyses suggested that cucurbitacin B caused G2/M arrest in human breast cancer cell lines (MDA-MB-231 and MCF-7) through elevated p21 expression [44]. Huang et al. showed that in human bladder cancer (T24) cells, cucurbitacin E-induced G2/M arrest was accompanied by a significant rise in p53 and p21 levels and a fall in the levels of STAT3, cyclin-dependent kinase 1 (CDK1) and cyclin B [45]. In addition, cucurbitacin E-induced G2/M phase arrest and death of T24 cells also depended on Fas/CD95 and mitochondria-dependent apoptotic pathways. Similarly, using other cancerous cell lines, cucurbitacins target the cell-cycle actions that involves growth inhibition, cell-cycle arrest at G2/M phase and induction of apoptosis [46]. Cucurbitacin I has been observed to suppress phosphotyrosine STAT3 in human cancerous lung cells [47]. Recently, it was observed to promote gastric cancer cell apoptosis by inducing the production of cellular ROS, as well as the endoplasmic reticulum stress pathway [40,48]. While cucurbitacin B, E and I have been observed to inhibit both JAK2 and STAT3 activation, cucurbitacin A and I have been reported to inhibit JAK2 and STAT3, respectively [47]. Treating Hep-2 cells with different concentrations of cucurbitacin B for various time intervals showed reduction in cell proliferation, cell-cycle distribution, and increased cell apoptosis in cancerous cell lines [46]. This study also stated that cucurbitacin B exhibited significant efficacy in inhibiting cell growth, arresting cell cycle at the G2/M phase, and inducing apoptosis in a dose-and time-dependent manner [46]. Similarly, cucurbitacins B, D, E were observed to inhibit proteins such as JAK-STAT3. They also inhibited mitogen-activated protein kinases (MAPK)-signaling pathways and tumor angiogenesis [48]. A study conducted on human umbilical vascular endothelial cell lines revealed cucurbitacin to significantly inhibit the proliferation, migration, and angiogenesis. It also blocked essential proteins such as Jak2-signal transducer, vascular endothelial growth factor receptor (VEGFR) and STAT3 signaling pathways [49]. Such studies have highlighted that the main mechanism involved in imparting the anti-tumorigenic potentials of cucurbitacins involves inhibition of the JAK/STAT3 signaling pathway, which plays an essential role in activation, proliferation, and maintenance of cancerous cells [14]. Another recent study has shown that treatment with 8 µM cucurbitacin IIb for 24 h remarkably inhibited the proliferation of HeLa and A549 tumor cells, with IC50 values of 7.3 and 7.8 µM, respectively, while increasing total apoptosis by 56.9 and 52.3%, respectively [50]. Another pathway by which cucurbitacin IIb induces apoptosis and cell-cycle arrest is by the regulating EGFR/MAPK pathway [51]. Similarly, cucurbitacin D was observed to regulate the levels of oncogenic signaling cascades, JAK/STAT, Wnt/β-catenin and associated non-coding RNAs in many cancer cell lines [52]. Recent studies have shown that CuIIb and cucurbitacin B induced apoptosis in cervical cancer cell lines by Nrf2 inhibition, whereas in lung cancer cell lines cucurbitacin B was responsible for suppressing growth and inducing apoptotic death by impeding IL-6/STAT3 signaling [21,53].
of cucurbitacins involves inhibition of the JAK/STAT3 signaling pathway, which plays an essential role in activation, proliferation, and maintenance of cancerous cells [14]. Another recent study has shown that treatment with 8 µM cucurbitacin IIb for 24 h remarkably inhibited the proliferation of HeLa and A549 tumor cells, with IC50 values of 7.3 and 7.8 µM, respectively, while increasing total apoptosis by 56.9 and 52.3%, respectively [50]. Another pathway by which cucurbitacin IIb induces apoptosis and cell-cycle arrest is by the regulating EGFR/MAPK pathway [51]. Similarly, cucurbitacin D was observed to regulate the levels of oncogenic signaling cascades, JAK/STAT, Wnt/β-catenin and associated non-coding RNAs in many cancer cell lines [52]. Recent studies have shown that CuIIb and cucurbitacin B induced apoptosis in cervical cancer cell lines by Nrf2 inhibition, whereas in lung cancer cell lines cucurbitacin B was responsible for suppressing growth and inducing apoptotic death by impeding IL-6/STAT3 signaling [21,53].

Antiangiogenic and Antimetastatic Mechanisms
The physiological process by which new blood vessels develop from pre-existing vessels is known as angiogenesis. Anti-angiogenesis causes suppression of tumor growth because of hunger and toxic waste buildup in its microenvironment [54,55]. The development and metastasis of the tumor have a major impact on the cancer vasculature ( Figure 3). Vascular endothelial growth factors (VEGFs) are crucial protein regulators of angiogenesis and metastasis. Studies have shown that inhibiting the VEGFR2-mediated JAK/STAT3 pathway is considered as an effective approach to suppress angiogenesis [49]. Though many studies about the mechanism of cucurbitacins and angiogenesis are not well known, few studies have still shown that cucurbitacins such as cucurbitacin B, cucurbitacin D, cucurbitacin E and cucurbitacin I possess anti-angiogenesis properties [56,57]. CuB significantly inhibited angiogenesis, metastasis, and vascular development in dose-dependent manner in in vivo models and chick embryos [56]. CuE significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation and angiogenesis by targeting the VEGFR2-mediated Jak2/STAT3 signaling pathway [49]. CuB has been observed to inhibit ERK1/2, prevent Raf-MEK-ERK from activating STAT3, which ultimately plays a key role in angiogenesis [58]. A similar effect of CuB was seen also in

Antiangiogenic and Antimetastatic Mechanisms
The physiological process by which new blood vessels develop from pre-existing vessels is known as angiogenesis. Anti-angiogenesis causes suppression of tumor growth because of hunger and toxic waste buildup in its microenvironment [54,55]. The development and metastasis of the tumor have a major impact on the cancer vasculature ( Figure 3). Vascular endothelial growth factors (VEGFs) are crucial protein regulators of angiogenesis and metastasis. Studies have shown that inhibiting the VEGFR2-mediated JAK/STAT3 pathway is considered as an effective approach to suppress angiogenesis [49]. Though many studies about the mechanism of cucurbitacins and angiogenesis are not well known, few studies have still shown that cucurbitacins such as cucurbitacin B, cucurbitacin D, cucurbitacin E and cucurbitacin I possess anti-angiogenesis properties [56,57]. CuB significantly inhibited angiogenesis, metastasis, and vascular development in dose-dependent manner in in vivo models and chick embryos [56]. CuE significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation and angiogenesis by targeting the VEGFR2-mediated Jak2/STAT3 signaling pathway [49]. CuB has been observed to inhibit ERK1/2, prevent Raf-MEK-ERK from activating STAT3, which ultimately plays a key role in angiogenesis [58]. A similar effect of CuB was seen also in human breast cancer cell lines. It successfully inhibited angiogenesis by targeting the FAK/MMP-9 signaling axis [59]. CuB showed antimetastatic activity and targeted angiogenesis also in paclitaxel resistant A2780/Taxol ovarian cancer cells. It also suppressed angiogenesis by downregulating the expression of HIF-1 targets, VEGF, VEGFR2 phosphorylation and erythropoietin [56,60]. Another study revealed the effective use of CuE for anti-angiogenesis in Huh7 cells. It decreased the tube formation in HUVECs and was also responsible for inhibiting the process of neo-vascularization in CAM assays [61]. A recent study showed that CuE modulated the JAK/STAT3 pathways, which regulated the angiogenesis [62]. CuE has been also involved in inhibiting the KDR/VEGFR2-mediated pathway of angiogenesis [63]. Treating A549 cells with cucurbitacins for~21 days showed positive results for inhibiting metastasis by regulating the levels of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D11 [64]. Similarly, other cucurbitacins were observed to inhibit angiogenesis in MDA-MB-231 and MCF-7 cancer cells by inhibiting the JAK/STAT pathways [65].
ogenesis also in paclitaxel resistant A2780/Taxol ovarian cancer cells. It also suppressed angiogenesis by downregulating the expression of HIF-1 targets, VEGF, VEGFR2 phosphorylation and erythropoietin [56,60]. Another study revealed the effective use of CuE for anti-angiogenesis in Huh7 cells. It decreased the tube formation in HUVECs and was also responsible for inhibiting the process of neo-vascularization in CAM assays [61]. A recent study showed that CuE modulated the JAK/STAT3 pathways, which regulated the angiogenesis [62]. CuE has been also involved in inhibiting the KDR/VEGFR2-mediated pathway of angiogenesis [63]. Treating A549 cells with cucurbitacins for ~21 days showed positive results for inhibiting metastasis by regulating the levels of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D11 [64]. Similarly, other cucurbitacins were observed to inhibit angiogenesis in MDA-MB-231 and MCF-7 cancer cells by inhibiting the JAK/STAT pathways [65].

Anti-Inflammatory Mechanisms
Most malignancies' growth and malignant progression are correlated with inflammation [66][67][68]. Both intrinsic and extrinsic inflammations have the potential to inhibit the immune system, which creates an ideal environment for the growth of tumors [69][70][71]. As a result, focusing on inflammation is a tempting strategy for both cancer therapy and cancer prevention [69,72]. Cucurbitacins have been observed to interact with proteins associated with inflammatory ( Figure 4) pathways such as interleukins (IL)-6, IL-5, IL-1β, IL-12, IL-13 in a dose-dependent manner [73]. Dietary cucurbitacin E has been shown to reduce inflammation and immunosuppression by downregulating the NF-κB signaling pathway [74]. CuB has been studied to possess protective effects by reducing inflammatory responses on sepsis-induced acute lung injury in in vivo rat models. It significantly

Anti-Inflammatory Mechanisms
Most malignancies' growth and malignant progression are correlated with inflammation [66][67][68]. Both intrinsic and extrinsic inflammations have the potential to inhibit the immune system, which creates an ideal environment for the growth of tumors [69][70][71]. As a result, focusing on inflammation is a tempting strategy for both cancer therapy and cancer prevention [69,72]. Cucurbitacins have been observed to interact with proteins associated with inflammatory ( Figure 4) pathways such as interleukins (IL)-6, IL-5, IL-1β, IL-12, IL-13 in a dose-dependent manner [73]. Dietary cucurbitacin E has been shown to reduce inflammation and immunosuppression by downregulating the NF-κB signaling pathway [74]. CuB has been studied to possess protective effects by reducing inflammatory responses on sepsis-induced acute lung injury in in vivo rat models. It significantly reduced the levels of TNF-α, IL-6, cytokine secretion and accumulation of inflammatory cells. It also regulated the levels of Ca 2+ , which play an essential role in inflammatory responses [75]. CuB inhibited inflammatory responses through targeting the SIRT1/IGFBPrP1/TGF β1 axis. It downregulated the expression levels of TGF β1, IGFBPrP1, and upregulated the expression of SIRT 1 [76]. Similarly, CuE decreased the levels of pro-inflammatory cytokines, such as IL-17 and IFN-γ, as well as the activities of the STAT3 and IL-17A-promoter in allo-reactive T cells [77]. CuE has been shown to inhibit skin inflammation and fibrosis by regulating the expression of α-Sma and Col-I in mice models [76]. Recently, it has also been demonstrated that CuE ameliorated lipopolysaccharide-evoked injuries and inflammation in bronchial epithelial cells by regulating the TLR4-NF-κB signaling. It was responsible for suppressing levels of inflammatory cytokine production, TNF-α, IL-6 and IL-8 [78].
Cucurbitacin B was observed to directly bind to toll-like receptor 4 (TLR4) and activate NLRP3 inflammasome, which further ultimately executed pyroptosis in A549 cells. CuB treatment has been observed to upregulate the protein expressions of IL-1β, GSDMD, HMGB1 and led to inhibition of generation of mitochondrial ROS and pyroptosis [79]. CuB was reported to sensitize CD133+ HepG2 cells in in vitro and in vivo models [80].
of pro-inflammatory cytokines, such as IL-17 and IFN-γ, as well as the activities of the STAT3 and IL-17A-promoter in allo-reactive T cells [77]. CuE has been shown to inhibit skin inflammation and fibrosis by regulating the expression of α-Sma and Col-I in mice models [76]. Recently, it has also been demonstrated that CuE ameliorated lipopolysaccharide-evoked injuries and inflammation in bronchial epithelial cells by regulating the TLR4-NF-κB signaling. It was responsible for suppressing levels of inflammatory cytokine production, TNF-α, IL-6 and IL-8 [78]. Cucurbitacin B was observed to directly bind to toll-like receptor 4 (TLR4) and activate NLRP3 inflammasome, which further ultimately executed pyroptosis in A549 cells. CuB treatment has been observed to upregulate the protein expressions of IL-1β, GSDMD, HMGB1 and led to inhibition of generation of mitochondrial ROS and pyroptosis [79]. CuB was reported to sensitize CD133+ HepG2 cells in in vitro and in vivo models [80].

Synergistic Effects with Other Drugs
During cancer therapies, the side effects and long-term consequences of anti-cancer chemotherapy continue to be a major cause of concern. The effectiveness of current medications to prevent the negative effects of chemotherapy is frequently insufficient. As a result, the current cancer treatment pattern is shifting toward combination chemo-

Synergistic Effects with Other Drugs
During cancer therapies, the side effects and long-term consequences of anti-cancer chemotherapy continue to be a major cause of concern. The effectiveness of current medications to prevent the negative effects of chemotherapy is frequently insufficient. As a result, the current cancer treatment pattern is shifting toward combination chemotherapy. A combination of medications improves the possibility that numerous oncogenic and resistance signalings will be inhibited simultaneously, whereas chemotherapeutics can affect cancer cells by affecting only one or two stages in the cell cycle. A modified cell line and the development of drug resistance are less likely when the malignant cells are attacked via several chemo-preventive agents. Recent research using the ovarian sarcoma M5076 cell line demonstrated that the synergism of cucurbitacin I and doxorubicin enhanced cytotoxicity and reduced the volume and weight of tumor cells. It was observed that treatment with CuI and doxorubicin decreased glutathione (GSH) levels, enhancing cytotoxicity in tumors. The phytochemical also increased DOX-induced antitumor activity [81]. In both in vitro and in vivo investigations, the combination of cucurbitacin B and curcumin proved particularly efficient against hepatocellular cancer. It encouraged apoptosis and reduced the potential for multidrug resistance in human hepatocarcinoma cells. Significant activity was observed with 2:1 ratio (cucurbitacin B:curcumin). It led to changes in tumor volume, caspase3 activation and ATP down-regulation, thereby serving as a novel, promising approach for treating human hepatoma [82]. Irinotecan and cucurbitacin have been found to have synergistic effects on the ability of colon cancer cell lines to resist proliferating, which together have increased their therapeutic benefits by activation of JAK2/STAT3, which plays a crucial role in cell survival and proliferation [83]. The synergistic effect of 23, 24-dihydrocucurbitacin B and cucurbitacin R was observed on inhibiting the expression of TNF-α, IL-6 through the NF-κB pathway in HepG2 cell line. Similar effects were observed with cucurbitacin D and docetaxel, which together effectively inhibited cancer cell growth and the cloning potential of prostate cancer stem cells. This combination has been suggested to be a novel therapeutic modality for the treatment of advanced prostate cancer [84,85]. Cucurbitacin B along with gemcitabine has been observed to induce apoptosis of MDA-MB-231 breast cancerous cells by regulating JAK/STAT3, Bcl-Xl, cyclin A and B1 [86], while with imatinib-mesylate it was responsible for inhibiting the proliferation of cells and inducing apoptosis through inhibition of MMP-2 expression [87]. Administration of cucurbitacin B and higenamine (in ratios 1:1, 1:2 and 2:1) significantly increased the cytotoxic effects on breast cancer cell lines, which also increased apoptosis and cell-cycle arrest in G2/M. This combination acts on essential proteins such as Akt farnesyl-transferase, platelet-derived growth factors, cyclin A2, CDK2, etc [88]. Cucurbitacin E with doxorubicin successfully induced apoptosis, cell-cycle arrest and autophagy [89]. Recent advances in the study of cucurbitacin IIb (CuIIb) and kinoin A (KinA) from Ibervillea sonorae (S.Watson) Greene highlight the ability of these molecules to reduce proliferation and to tempt apoptotic and cell-cycle apprehension in tumors; also, the levels of STAT3 expression were downregulated after treatment with CuIIb. These findings imply that CuIIb and KinA may be considered in future research for the creation of efficient and secure anti-cancer treatments for breast, cervical, gastric and other cancer types where STAT3 is overexpressed [90].

Safety Studies
As several researchers have identified the presence of toxic cucurbitacins in the roots, leaves, and fruits of some plants, such as Combretum zeyheri Sond and Cucumis anguria L. [91], further studies are highly needed to evaluate the safety issue and determine the value of the no-observed-adverse-effect-level (NOAEL), before any pharmacological applications of cucurbitacins as anticancer agents can be recommended. On the other hand, it has been claimed that safety issues of cucurbitacins may be related to their specific variants, the purity of preparations and differences in the study models [92]. For example, no toxic reactions were observed in lung cancer xenografted mice treated intraperitoneally with 1 mg/kg of cucurbitacin B [93] or cucurbitacin Q [94]. Today, it is generally accepted that the active dose and lethal dose are not the same for different types of cucurbitacins, being related to their structural peculiarities [92]. Therefore, further clinical trials on the safety and efficacy of specific variants of cucurbitacins are highly required. Tables 1 and 2 present an outline of diverse antiproliferative actions of cucurbitacins.

Conclusions and Further Perspectives
As demonstrated in the present review article, diverse variants of cucurbitacins can be active against different types of malignancies. Moreover, the combination of cucurbitacins with conventional chemotherapeutic drugs might lead to synergistic anticancer effects, revealing a great promise for the application of these compounds in future clinical settings as either individual agents or drug adjuvants. However, many steps have remained to achieve this attractive goal. First, the safety of cucurbitacins must be elucidated, jointly with their metabolic conversion and possible bioactivities of various metabolites. Secondly, the proper dosage regimens also need to be elaborated. Studies related to the pharmacokinetic properties of cucurbitacins, including t1/2, Cmax, Tmax, Vd, mean residence time, etc., should be carried out singly or in synergism. Investigations on the metabolic conversions of cucurbitacins will improve its bioavailability and stability prospective. In this way, the current review presents a strong basis to move on with these next steps in the path to ultimately find novel, safe and more efficient therapies against cancer.