Elsevier

Phytomedicine

Volume 92, November 2021, 153737
Phytomedicine

Anticancer effects of asiatic acid against doxorubicin-resistant breast cancer cells via an AMPK-dependent pathway in vitro

https://doi.org/10.1016/j.phymed.2021.153737Get rights and content

Abstract

Background and purpose

Asiatic acid is one of the active compounds isolated from Centella asiatica and has been used to treat many diseases, including hypertension, pulmonary fibrosis, and cancer. It exhibits anticancer effects in many cancers, such as ovarian, lung and colon cancer; however, its anticancer effects in breast cancer and the underlying mechanism are not fully understood. Chemoresistance is often induced after the use of chemotherapy, and it is a challenging problem in cancer therapy. The effects of asiatic acid on chemoresistance in breast cancer have never been studied. Therefore, the aim of the present study was to examine the anticancer effects of asiatic acid in doxorubicin-resistant breast cancer MCF-7 cells.

Methods

The cells were incubated with asiatic acid at 0-160 μM for 2-24 h. Cell viability and cytotoxicity were evaluated by 3-[4, 5-dimethyl-2-thiazolyl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Florescent images were taken using a confocal microscope. P-gp function and apoptosis assays were performed using flow cytometry. Caspase activity was measured with the Caspase-Glo™ Assay System. The phosphorylation and expression of relevant proteins were assessed by western blots. Molecular docking was performed and scored by AutoDock. Cellular thermal shift assay (CETSA) was applied for experimental valuation.

Results

Our data demonstrated that asiatic acid induced cell death in multiple ways, including reactive oxygen species production, adenosine triphosphate (ATP) content reduction, and adaptive immunity balance via intrinsic apoptosis, AMP-activated protein kinase (AMPK), programmed death-ligand 1 (PD-L1), and indirect nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional pathways, using experimental validation and in silico analysis. Moreover, asiatic acid also enhanced the sensitivity of doxorubicin-resistant MCF-7 cells to doxorubicin by improving P-glycoprotein (P-gp) function.

Conclusions

This study provides evidence that asiatic acid has strong anticancer effects to reverse multidrug resistance and could be developed as a promising adjuvant drug for the treatment of chemoresistant cancer.

Introduction

Breast cancer is one of the most common cancers worldwide. According to Cancer Statistics 2020 in the United States, breast cancer is the most common cancer for women (30% of new diagnoses in female cancer), and the greatest number of female cancer deaths is from breast cancer (15% of female cancer deaths) (Siegel et al., 2020). Metastasis is one of the main causes of death in breast cancer, where tumor cells proliferate, reorganize, and migrate into other tissues (Borin et al., 2016). Chemotherapy is an adjuvant therapy after the removal of tumours by surgery, and it is also used to prevent and inhibit metastasis (Anampa et al., 2015; Lee et al., 2017; Zuo et al., 2019). However, drug resistance often occurs after chemotherapy, which leads to the worsening of cancer treatment and shows broad correlations in AMP-activated protein kinase (AMPK)-mediated metabolic pathways (Mansoori et al., 2017; Tan et al., 2019). Doxorubicin (DOX) is a commonly used chemotherapeutic drug for breast cancer, while DOX resistance largely reduces the efficacy of chemo-based drugs (Wijdeven et al., 2015). Therefore, improving sensitivity to DOX is one of the main strategies in treating breast cancer through modulating the expression and function of ATP-binding cassette (ABC) transporters, including P-glycoprotein 1 (P-gp) and multidrug resistance-associated protein 1 (MRP1) (Datta et al., 2015).

The use of chemotherapy results in the induction of cancer cell death with morphological characteristics of apoptosis (Doonan and Cotter, 2008). There are two main apoptotic pathways, intrinsic and extrinsic pathways, and targeting these pathways is one of the main strategies for cancer therapy (Baritaki et al., 2011; Jensen et al., 2008). Intracellular signals that are regulated by mitochondria are involved in intrinsic pathways. These signals could induce the loss of mitochondrial membrane potential and cytochrome c release, which in turn leads to caspase-9 and caspase-3 activation, and cell death (Du et al., 2000). On the other hand, the extrinsic pathway is mediated by transmembrane ligand-gated death receptors, such as the tumour necrosis factor (TNF) receptor (Ashkenazi and Dixit, 1998). These death receptors activate caspase-8 and eventually lead to caspase-3 activation and cell death (McIlwain et al., 2015).

Centella asiatica (L.) is a phytomedicine that is used as a culinary vegetable and as a medicinal herb. As a popular skincare product in cosmetology, the extract of C. asiatica (L.) is widely used in wound healing and the treatment of psoriasis and scleroderma, and is also applied to photoaging skin, cellulite, and striae. As a medicinal use, it has been used to treat many diseases, including cancer, cerebral ischaemia, and hypertension (Hsu et al., 2005; Krishnamurthy et al., 2009; Meng et al., 2019), and has many biological effects, such as anti-inflammatory, anticancer, and antioxidative activities (Lv et al., 2017; Sakonsinsiri et al., 2018). Previous studies demonstrated that the extract of C. asiatica (L.) exhibited anticaner effects in various cancers including lung and breast cancer, melanoma, glioma, and hepatoma (Babykutty et al., 2008; Lin et al., 2002; Naidoo et al., 2017; Pittella et al., 2009). Asiatic acid, a pentacyclic triterpene derivative, is an active compound isolated from C. asiatica (L.) and exhibits strong anticancer effects on breast cancer cells (Hsu et al., 2005); however, the underlying mechanisms are not fully understood. Moreover, its effect was not studied in chemoresistant breast cancer cells, so our present study aimed to investigate the novel effects of asiatic acid in DOX-resistant MCF-7 breast cancer (MCF-7/DOXR) cells and their underlying mechanisms.

Section snippets

Cell culture and drug treatment

The MCF-7 cell line was obtained from American Type Culture Collection (ATCC; Manassas, USA), and the MCF-7/DOXR cell line was established by stepwise exposure of MCF7 cells to increased concentrations of DOX as described previously (Zhong et al., 2016). The cells were cultured with RPMI 1640 medium containing fetal bovine serum (FBS, 10%) and penicillin-streptomycin (100 units/ml), equilibrated with 5% CO2 and 95% air at 37 °C. Asiatic acid (≥ 98%; Sigma-Aldrich, USA) and DOX (98.0-102.0%;

Asiatic acid induced cell death in MCF-7/DOXR and MCF-7/DOXS cells

Fig. 1A and B show that the plant of C. asiatica (L.) and the chemical structure of one of its bioactive compounds, asiatic acid, respectively. Treatment iwth asiatic acid for 24-h dose-dependently inhibited MCF-7/DOXR cell viability (Fig. 1C). At 40, 80 and 160 μM, it significantly inhibited cell viability, and the IC50 value was 89.06 μM. In addition, asiatic acid also significantly induced LDH release in MCF-7/DOXR cells to 261.15% and 506.11% at 80 and 160 μM, respectively (Fig. 1D).

Discussion

Chemoresistance is a challenging problem for chemotherapy, and DOX resistance commonly occurrs in breast cancer treatment (Wijdeven et al., 2015); therefore, natural agents that target ABC transporters to enhance the sensitivity of DOX are potential drugs for breast cancer treatment (Luo et al., 2019). There is growing evidence showing that asiatic acid has anticancer effects in various cancers, including ovarian, lung and colon cancers, cholangiocarcinoma, hepatocellular carcinoma, and

Conclusions

The present study demonstrated the anticancer effects of asiatic acid in chemoresistant breast cancer cells. We showed that asiatic acid effectively induced cell death and apoptosis to reverse DOX resistance in multiple ways, including reactive oxygen species generation, ATP content reduction, P-gp function improvement, and adaptive immunity balance via intrinsic apoptosis, AMPK, and indirect NF-κB transcriptional pathways, using experimental validation and in silico analysis. Taken together,

Authors' contribution

ZZ and ZZF designed and conducted experiments. YJ and HYJ performed the in-silico analysis. VCT analysed data and drafted the manuscript. XJB and CG helped to coordinate the study. CL and HZW organized and provided valuable feedback to our study. All authors read and approved the manuscript. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Funding

This study was supported by the Macao Science and Technology Development Fund (014/2017/A1), the Research Fund of University of Macau (CPG2021-00022-ICMS), Special Fund of Guangdong Science and Technology Plan in 2016 (2016B030309002), the China Postdoctoral Science Foundation funded Project (2017M622811), the Natural Science Foundation of Guangdong Province, China (2018A030310226 and 2020A1515010922), the Educational Commission of Guangdong Province, China (2017KQNCX084), and Traditional

Declaration of Competing Interest

There are no potential conflicts of interest to declare.

Acknowledgment

Special thanks to Mr. Wenmin Zhong for taking picture of the plant of C. asiatica (L.) in the villages of Guangdong province, China.

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