Effect of a Thai Herbal Recipe, Benja Amarit, on induction of human cancer cell death

Abstract

Liver cancer and colorectal cancer are two of the five most common cancers and are the most common causes of cancer death globally. In Thailand, cholangiocarcinoma (CCA) is the predominant pathological type of primary liver cancer that is the highest incidence and prevalence of the world. At the present, there are many techniques for cancer treatment includes surgery, chemotherapy, radiotherapy as well as immunotherapy. Most of cancer patients are prescribed chemotherapy due to their frequently late diagnosis. However, the patients always suffer from their side effects and the increasing prevalence of cancer resistance, which requires further research to develop the efficient therapy without adverse effects. Benja Amarit (BJA) recipe is a traditional herbal formula which contains nine ingredients. It has been used for treating diseases or symptoms that relate with liver and colon as well as cancer treatment for more than a century in Thailand. However, there has been no evidence-based research to support the antitumor activity of this recipe along with the mechanisms of action that are involved. In this study the anticancer potential of BJA recipe on human hepatocellular carcinoma, cholangiocarcinoma and colon cancer cells have been investigated. The BJA recipe was extracted by 95% ethanol (BJA-95), 50% ethanol (BJA-50) and water (BJAW), then the cytotoxic effects of the extracts were determined by using thiazolyl blue tetrazolium bromide (MTT) assay. Interestingly, BJA-95 and BJA-50 significantly inhibited cancer cell viability in a dose-dependent manner, especially on high-invasive cholangiocarcinoma KKU-213 cells with IC50 values at 7.1±1.3 μg/mL and 11.1±1.5 μg/mL and on hepatocellular carcinoma HepG2 cells with IC50 values at 9.1±2.4 μg/mL and 27.2±4.1 μg/mL, respectively. However, BJA-95 was found to be the most effective extract by presenting the lowest IC50 values in all cancer cell lines. Meanwhile, this extract was more toxic to KKU-213 and HepG2 cells than towards normal cell lines and blood cells. Hence, the most effective extract (BJA-95) and the most sensitive cancer cell lines (KKU-213 and HepG2) were selected for subsequent experiments. The phytochemical constituents of BJA-95, BJA-50 and BJA-W were analysed by using gas chromatography–mass spectrometry (GC-MS) and liquid chromatography– mass spectrometry (LC-MS). The results revealed that BJA extracts consistes of fortyeight different compounds. While piperine, xanthotoxol and dihydrogambogic acid were the main compounds that mostly were found in the highest effective extract BJA-95, followed by BJA-50, while less found or absence in non-effective extract (BJA-W). The mechanisms of action of BJA extract through apoptosis induction were investigated and quantitated by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining employing flow cytometry. The result showed that BJA-95 caused both KKU-213 and HepG2 cells to undergo apoptosis. Mechanistically, the extract increased the expression levels of pro-apoptotic Bax and Puma proteins, while the expression levels of anti-apoptotic Bcl-xL protein was reduced. It also increased the activity of caspase-3, -8 and -9 which were determined by specific substrates with colorimetric analysis and increased the percentage of cells with the loss of mitochondrial transmembrane potential (ΔΨm), which were measured by specific fluorescence probes DiOC6 (3,3’-dihexyloxacarbocyanine iodide). It indicated that BJA-95 induced KKU-213 and HepG2 cancer cells apoptosis via mitochondrial and caspase-dependent pathways. The mechanisms of action of BJA extract upon autophagy was determined by specific fluorescence dye then the autophagic vacuoles was imaged under fluorescence microscopy and quantitated by flow cytometry. The result showed that BJA-95 induced autophagy only in KKU-213 cells, while autophagic flux did not appear within HepG2 cells. Mechanistically, the extract significantly increased the expression levels of autophagic-related proteins including Baclin-1, ATG5 and LC3-II. However, autophagic response to therapy not only leads to cell death, it also acts in stress clearing and maintaining for cell survival. Thus, the cancer cell viability was determined under BJA- 95 treatment with or without autophagy inhibitor (3-methyl adenine, 3-MA). Intriguingly, co-treatment of 3-MA could reduce KKU-213 cell viability more then BJA-95 treatment alone, which indicated that autophagic response to BJA extract played a role in maintaining the KKU-213 cell survival. Moreover, the results showed that inhibition of autophagy by 3-MA could enhance BJA-95 induced-KKU-213 cells death via apoptosis. This antitumor potential of BJA-95 was confirmed in an in vivo model by using xenograft model. The KKU-213 cells were inoculated subcutaneously in nude mice and then the mice were treated with BJA-95 at 25 and 50 mg/kg for 20 days. The results showed that the tumor volume and tumor weight in treatment groups were significantly lower and different from the control group. Meanwhile, the extract did not affect animal body weight and it was found to be non-toxic to blood cells, kidneys, and the liver. The resulted fibrosis was originated from cancer cells apoptosis within tumor tissue was increased in treatment groups and almost 80% of the viable tumor mass was reduced when compared to the control, which exhibited by hematoxylin and eosin (H&E) staining. Additionally, immunohistochemistry (IHC) revealed that BJA-95 increased the expression levels of pro-apoptotic Bax and active caspase 3 proteins in tumor tissue, while the expression levels of anti-apoptotic Bcl-xL protein was reduced. It indicated that BJA- 95 inhibited tumor growth in an in vivo by targeting apoptosis. In conclusion, BJA extract exhibited the high effectiveness against liver cancer both hepatocellular carcinoma and particularly high-invasive cholangiocarcinoma without discernible side effects. Moreover, efficacy of BJA against cholangiocarcinoma could be enhanced by combination treatment with autophagy inhibitor (3-MA), which will be of high potential and reserved strategy in treatment of the future chemotherapeutic resistance. Therefore, BJA extract is considered as an alternative therapy for liver cancer patients, both hepatocellular carcinoma and cholangiocarcinoma.