Background

It is estimated that fatalities arising from pancreatic cancer will be second only to lung cancer by 2030. Overall 5-year survival rates for pancreatic cancer have remained unchanged for decades and patient prognosis remains poor despite efforts to better understand the tumor microenvironment [1,2,3]. Therefore, novel treatment strategies for pancreatic cancer are in dire need. Current chemotherapeutic regimens used in treating pancreatic cancer suffer from debilitating side effects which limit tolerable doses [4]. Furthermore, many therapies exacerbate the mutation of cancers to metastatic and drug resistant phenotypes. Yet, the majority of modern cancer therapeutics are derived from plants [5]. Concerning anti-tumor effects, previous studies have reported that various natural products can demonstrate anticancer efficacy through inhibition of cell growth [5, 6].

Tetrastigma hemsleyanum Diels et. Gilg (Sanyeqing) is a rare, endangered medicinal plant native to China and traditionally used in folk medicine to treat a variety of cancers. Extracts from its root tuber, stems, leaves or whole plant are proven to be effective against various types of cancer [6,7,11, 12]. Characterized by its aggressive biological nature and strong resistance against chemotherapeutic treatment or radiation therapy, the 5-year survival rate of pancreatic cancer has shown little improvement in the past decade at < 5% survival [1, 3, 11,12,13]. Because chemotherapy and radiation therapy produce detrimental side effects, an urgent need for treatment strategies has arisen for pancreatic cancer. Recently, traditional Chinese medicines have garnered increasing attention due to their potential as anti-tumor agents. The majority of traditional Chinese medicines are derived from plant species, which have been used against cancer for centuries [5, 14, 15]. Yet, mechanisms underlying their efficacy remain unclear. Current research suggests that the mechanism of action occurs through cell cycle arrest, induction of apoptosis and inhibition of migration [8, 15,16,17]. Previous studies have shown that an ethyl acetate fraction of Tetrastigma hemsleyanum (EFT) can cause cell cycle arrest and induce apoptosis in human hepatoma HepG2 cells [7]. In this study, the underlying antitumor mechanisms were profiled in PANC-1 human pancreatic cancer cells treated with EFT.

Cell cycle control describes the regulatory process of cell growth that is dictated by coordinated interactions of various cyclins and their respective cyclin-dependent kinases (CDKs) [18, 19]. Cyclins and CDKs together form various checkpoints that can either usher or inhibit progression of a cell through the cell cycle. If the key steps at each regulatory phase of the cell cycle are completed, cyclin/CDK interactions allow progression into the next phase of the cycle [18,19,20]. However, prolonged arrest at a particular checkpoint can occur due to genetic instability or mutation; both key contributors to cancer development [20,21,22]. Not surprisingly, a variety of anticancer agents arrest the cell cycle at particular checkpoints to induce apoptosis in cancer cells [16, 21]. As seen from Fig. 3, S phase arrest was induced by EFT in a dose-dependent manner through downregulation of CDK2. This result indicates that the mechanism of EFT cancer therapy is, in part, due to the downregulation of CDK2 that induces cell cycle arrest and subsequent apoptosis. Yet, the mechanism of EFT is not solely limited to apoptosis. EFT has also shown potential to prevent migration and invasion of PANC-1 cells in a dose-dependent manner in Fig. 4. Since pancreatic cancer is characterized by excessive migration and invasion, methods to specifically inhibit or reverse these malignant features are crucial in both basic and preclinical research [22,23,24]. Largely, EFT’s anti-metastatic properties can largely be related to the epithelial to mesenchymal transition (EMT) and matrix metalloproteinases (MMPs).

EMT is a biological process where cells lose epithelial characteristics and obtain a mesenchymal phenotype, which improves cell motility by decreasing cell-cell adhesion [24]. Many studies point to the activation EMT as the critical mechanism for the acquisition of malignant phenotypes in epithelial cancer cells [24,25,26,27]. EMT is based on the down-regulation of epithelial markers such as E-cadherin while mesenchymal markers, such as ZEB1, Vimentin, N-cadherin and Fibronectin, are up-regulated [28]. Therefore, EMT or mesenchymal properties are an ideal target for cancer therapy.

MMPs are a family of zinc-dependent endopeptidases essential for extracellular matrix degradation [29]. Among all MMPs, MMP-2 and MMP-9 are key to basement membrane type IV collagen degradation during cancer progression. Particularly MMP-2 and MMP-9 are well known for promoting tumor migration and invasion [30, 31]. Aside from direct regulation of basement membrane degradation, cross talk between MMPs and EMT can regulate the invasion or migration of cancer cells. It has been demonstrated that overexpression of MMP-2 or MMP-9 led to induction of EMT in breast cancer [32]. Yet, a variety of EMT processes strengthen MMP expression in return, suggesting an intricate positive feedback loop between MMPs and EMT that synergistically contributes to migration and invasion in malignant tumors.

Therefore, strategies inhibiting both EMT and MMPs are an efficient approach to cancer therapy. As seen from Fig. 5, downregulation of mesenchymal markers such as ZEB1, Vimentin, N-cadherin and Fibronectin and upregulation of epithelial markers such as E-cadherin demonstrate the reversal of the metastatic EMT phenotype induced by EFT. Furthermore, downregulation of MMP-2 and MMP-9 were also identified after EFT treatment. These results suggest that EFT interrupts the positive feedback loop between EMT and MMPs by downregulating key cytokines known for EMT while also downregulating MMPs. These results further contribute to the anti-invasion properties found of EFT against PANC-1 human pancreatic cancer cells.

In summary, pancreatic cancer remains one of the most lethal cancers with 5% of 5-year survival rate. Its poor prognosis is mainly attributed to the nature of the cancer which is both drug resistant and highly metastatic. For treatment of pancreatic cancer, EFT has been identified as a potent therapeutic. As seen from Table 1, GC - MS analysis has revealed that the chemical constituents of EFT are primarily organic acids and their esters, among which succinic acid (10.56%) [33, 34] and benzoic acid (8.65%) had antioxidant, anti-inflammatory effects and had anti-tumor potential. Fatty acids [35,36,37] including linoleic acid (14.37%) and 9,12,15-all-cis-Octadecatrienoic acid (9.46%) have shown activity in cancer prevention and treatment. In recent years, a number of studies have shown that palmitic acid has become a promising antineoplastic agent with demonstrated effects on various malignancies including stomach cancer, liver cancer, cervical cancer, breast cancer and colorectal cancer [38,39,40,41,42,43]. The anti-tumor effects include inducing apoptosis of tumor cells, inhibiting proliferation of tumor cells, inhibiting metastasis and invasion, increasing sensitivity to chemotherapy, and improving immune function. Our findings suggest that EFT contains high levels of palmitic acid (12.02%) and may therefore have the same potential against tumors.

While EFT has shown potent efficacy against PANC-1 cells in vitro, its mechanism of efficacy is poorly understood. Studies performed herein demonstrated that EFT demonstrates significant cytotoxicity against PANC-1 through a cytotoxicity assay. Furthermore, EFT was found to inhibit PANC-1 migration through a wound healing assay. It was determined that EFT inhibits progression of PANC-1 through a collection of key properties. Firstly, EFT halts PANC-1 growth by inducing S-phase cell cycle arrest through downregulation of CDK2. Secondly, EFT tackles the issue of PANC-1 invasion and metastasis through a two-pronged approach. By increasing expression of cell-cell adhesion molecules, EFT substantially reverses the EMT, a key contributor to metastasis. Furthermore, EFT acts to downregulate MMP-2 and MMP-9 which are primarily known for inducing basement membrane degradation. By strengthening the basement membrane and promoting cell-cell adhesion, EFT interrupts the positive feedback loop between EMT and MMPs that are known contributors to the metastasis of PANC-1 cells. Based on the results of this study, the mechanisms by which EFT affects PANC-1 cells are summarized in Fig. 6.

Fig. 6
figure 6

The proposed mechanism of EFT-induced S phase cell cycle arrest, inhibition of migration and invasion of PANC-1 cells

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Conclusion

This study confirmed the anti-cancer properties of EFT on human pancreatic cancer PANC-1 cells. EFT exhibits efficacy through multiple avenues; such as inhibition of cell growth, inhibition cell invasion, cell cycle arrest, inhibition of mesenchymal properties and MMPs. In conclusion, these results affirm that Tetrastigma hemsleyanum serves as a valuable therapeutic for the treatment of pancreatic cancer.