Cancer is one of the most common life-threatening diseases in the 21st century with rising incidence rates and progressively reducing age of the afflicted patients. There are evidences that inflammation and cancer development has strong interaction [1]. RCC is the most lethal malignant tumor of the kidney and exhibits highly vascularized and metastatic characteristics. RCC is the most common malignancy (about 85%) of the kidney and accounts for approximately 3% of adult cancers. The selection of 786-O cells in this study was therefore based on the clinical and epidemiological data of RCC as well as their specific cellular properties. Thus, 786-O cells are considered to be an appropriate model for RCC and have been widely used in different types of RCC research such as proliferation, apoptosis, migration and invasion, metastasis or therapy resistance studies [31–33].
In addition to analyzing HWG, RA and CDDP toxicity in normal cells, we exposed HK-2 cells to the same concentrations as 786-O cancer cells. HK-2 cells were obtained from a primary proximal tubular cell culture of the normal adult human renal cortex and exposed to a recombinant retrovirus containing HPV 16 E6/E7 genes. These cells retain a phenotype indicative of well-differentiated primary proximal tubular epithelium and retain functional characteristics of proximal tubular epithelium [34]. Interestingly, HWG and RA displayed a very low efficiency in HK-2 cells, indicating that HWG and RA are less toxic for normal cells than for 786-O cancer cells. On the contrary, CDDP highly reduced the growth of 786-O cancer cells than HK-2 cells.
Our study shows that HWG, RA and CDDP dose-dependently inhibit HK-2 and 786-O cell proliferation. Meanwhile, CDDP showed toxicity on human RCC 786-O cells. Since reports of CDDP’s cytotoxic effect vary across studies and may depend on the cell types, our study used two cell types HK-2 and RCC 786-O cells. The concentration of CDDP that reduced cell viability by about 50% compared to RCC 786-O cells was less than that of HK-2 cells, indicating RCC 786-O cells are more sensitive to CDDP. Thongnuanjan et al., reported that CDDP is transported into renal proximal tubular cells via the renal organic cation transporter 2 (OCT2) but not so in HK-2 cells [35].
Chinese medicines have been widely used as health-promoting food ingredients and supplements for the treatment of cancer. The combination of anti-tumor agents should be able to produce synergistic or additive therapeutic efficacies, reduced side effects and minimal drug resistance. Experimental strategies that investigate combinational therapy of CDDP with natural plant extracts/plant-derived agents to maintain the desired antitumor efficacy have been reported [9, 36, 37]. In this study, HWG in combination with CDDP inhibits the invasion of these RCC cells, especially at a concentration of more than 100 µg/mL and inhibited the wound-healing migration of these RCC cells, especially at a concentration of more than 200 µg/mL. RA in combination with CDDP inhibited the invasion of these RCC cells, at a concentration of more than 25 and 50 µM. But, RA in combination with CDDP did not inhibit the wound healing migration of these RCC cells. Here, we demonstrate the preventive and therapeutic effect of HWG and RA alone as well as their synergism with CDDP, against RCC 786-O metastasis. Rosmarinic acid’s anti-cancer activity was not restricted to the colon, but also included skin cancer and melanoma, pancreatic cancer, breast cancer, lung cancer, leukemia, hepatoma and ovarian cancer [21]. Fisetin (3,3’,4’,7-tetrahydroxyfavone), a naturally occurring flavonoid commonly found in plants is effective against cancer, and its possible mechanisms includes suppression of proliferation and metastasis of RCC through upregulation of MEK/ERK-Targeting CTSS and ADAM9 [32].
Our in vitro mechanistic study shows that HWG/CDDP and RA/CDDP synergistically inhibit or disrupt RCC 786-O cell motility and cell-cycle machinery. Thus, HWG and RA inhibit 786-O cell proliferation, as well as arrest cell cycle in the G2/M phase. Induction of apoptosis by HWG and RA, and in combination with CDDP was assessed in 786-O cells through treatment with the extract/compound followed by flow cytometric analysis. We conducted an Annexin V-FITC/PI double staining assay to investigate whether HWG/CDDP and RA/CDDP combination reduced RCC 786-O cells viability via apoptosis induction. Only RA in combination with CDDP induced significantly less apoptosis at 48 h. Several reports have previously described that CDDP induces S- and G2/M-phase arrest in a sequential manner [38]. Apoptosis is an essential cell process in the homeostasis of multicellular organisms, and its dysregulation has been involved in many human tumors [39, 40]. G2/M phase cell cycle arrest is one of the most prominent checkpoints of many anticancer agents which can reduce proliferation and then induce apoptosis by inhibiting the segregation of damaged chromosomes during mitosis [41–43].
CDDP induces apoptotic cell death as evidenced by increased cleavage of caspase-3 and PARP. During apoptosis, cleavage of PARP by caspase-3 in response to DNA strand breaks has become a hallmark of this type of cell death. Our data indicates that HWG and RA and their combination with CDDP significantly induces apoptosis by PARP. Thus, the protein levels of cleaved-PARP and PARP were assessed by Western blot analysis. The data demonstrates that cisplatin induced high expressed of cleaved-PARP compared with the control group. PARP-1 is a poly-(ADP-ribosylated) enzyme necessary for DNA repair. Thus, cleaved PARP-1 is considered to be a remarkable marker of apoptosis. PARPs modify target proteins post-translationally with poly (ADP-ribose) (PAR) or mono (ADP-ribose) (MAR) using NAD+ as substrate, and play a central role in renal epithelial cell apoptosis during cisplatin nephrotoxicity [44, 45].
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays a vital role in focal adhesion. It is regulated by multiple phosphorylation sites, tyrosine 397, Tyr 576/577, and Tyr 925. Wound healing experiments were done to evaluate how Tyr-925 phosphorylation affects cell migration. FAK activation, results in FAK phosphorylation at Tyr-397 (Tyr(P)-397), promotes Src family protein-tyrosine kinase binding to the FAK Tyr(P)-397 site, and facilitates the formation of the FAK-Src signaling complex that results in the secondary phosphorylation of FAK at Tyr-861 and Tyr-925. FAK, a protein tyrosine kinase, is overexpressed in several cancers and promotes cancer progression and metastasis [46, 47]. Our Western blot result demonstrated that RA in combination with CDDP significantly inhibited the expressions of p-FAK (Tyr 925) in RCC 786-O cell, not observed in HWG.
Cell migration is implicated in various processes including embryogenesis, tissue regeneration, wound healing, and tumor progression. During this process, cells interact with the microenvironment in part through focal adhesions. FAK clusters at focal adhesion structures regulates cancer-associated processes, including adhesion, migration and invasion [48, 49]. Moreover, cell cycle analysis revealed that FAK inhibition induced G1 arrest in 786-O cells. On the basis of these results, we propose that the inhibition of RA on RCC 786-O cell invasion and migration may partly occur through the downregulation of FAK phosphorylation.
RA is a highly valued natural phenolic compound that is very commonly found in plants families Lamiaceae and Boraginaceae. In a review of the biological effects of RA, Moore et al. concluded that it could be used as a phytochemical to induce apoptosis. RA can reduce survival of cancer cell lines such as HT-28, MCF-7, DU145 or MKN45, among others. The administration of RA in colon cancer cell lines (HT-29) reduced a transcription factor through inhibition of activator protein-1 (AP-1), which is responsible for the activation of COX-2. The anti-cancer activity of RA was not restricted only to the colon, but also included skin cancer and melanoma, pancreatic cancer, breast cancer, lung cancer, leukemia, hepatoma and ovarian cancer [21, 50, 51].
Organic compounds that have been discovered in natural sources, such as plants, animals, and microorganisms, have been an inspiration for drug development. Newman and Cragg concluded that more than 50% of all modern clinical drugs have their origin in natural products [52]. Phytochemicals exert antitumor effects via distinct mechanisms. They selectively kill rapidly dividing cells, target abnormally expressed molecular factors, remove oxidative stress, modulate cell growth factors, inhibit angiogenesis of cancerous tissue, and induce apoptosis. For example, some polyphenols (resveratrol, gallocatechins), flavonoids (methoxy licoflavanone, alpinumisoflavone), and brassinosteroids (homocatasterone and epibrassinolide) exert anticancer effects through apoptosis induction [53]. Combination of CDDP with phytochemicals can augment the anti-cancer activity of CDDP by triggering apoptosis. In our previous study, RA was the most abundant phytochemicals in G. hederacea. But, RA and HWG combination treatment with CDDP on RCC 786-O cells have not been consistent. Therefore it might be other pharmaceutical and nutraceutical components in HWG that would be effective. These results suggest that RA is the major active compound responsible at least in part for the anticancer effect of HWG. We further proceeded to identify the active compound responsible for the cytotoxic activity of the extract by an activity-guided fractionation approach. Our study provides a novel insight on the synergistic use of HWG and RA, natural plant products as chemosensitizers, and cisplatin in the management of 786-O cells. Plant-derived anticancer drugs/natural products are an important resource in the discovery of lead compounds for anti-cancer drug development.