Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.

Hypoxia-inducible factor-1α (HIF-1α) is a major transcriptional regulator that plays a crucial role in the hypoxic response of rapidly growing tumors. Overexpression of HIF-1α has been associated with breast cancer metastasis and poor clinical prognosis. Plumbagin, the main phytochemical from Plumbago indica, exerts anticancer effects via multiple mechanisms. However, its precise mechanisms on breast cancer cells under hypoxic conditions has never been investigated. This study aims to examine the anticancer effect of plumbagin on MCF-7 cell viability, transcriptional activity, and protein expression of HIF-1α under normoxia and hypoxia-mimicking conditions, as well as reveal the underlying signaling pathways. The results demonstrate that plumbagin decreased MCF-7 cell viability under normoxic conditions, and a greater extent of reduction was observed upon exposure to hypoxic conditions induced by cobalt chloride (CoCl₂). Mechanistically, MCF-7 cells upregulated the expression of HIF-1α protein, mRNA, and the VEGF target gene under CoCl₂-induced hypoxia, which were abolished by plumbagin treatment. In addition, inhibition of HIF-1α and its downstream targets did not affect the signaling transduction of the PI3K/Akt/mTOR pathway under hypoxic state. This study provides mechanistic insight into the anticancer activity of plumbagin in breast cancer cells under hypoxic conditions by abolishing HIF-1α at transcription and post-translational modifications.

Colorectal cancer is the second leading cause of cancer-related mortality worldwide, highlighting the critical need for novel therapeutic strategies. In this study, we investigated the anticancer activity and molecular mechanisms of RS-PP-059, a derivative of 14-deoxy-12-hydroxyandrographolide, in colorectal cancer cells. RS-PP-059 exhibited potent cytotoxicity and selectivity toward HT-29 cells, suppressing viability and clonogenic growth. The compound induced apoptotic cell death, as shown by increased Annexin V-positive cells, PARP-1 cleavage, p53 activation, and γ-H2AX accumulation, indicating DNA damage, and was accompanied by a reduction in total caspase-3 protein levels. Mechanistically, RS-PP-059 triggered endoplasmic reticulum (ER) stress and unfolded protein response (UPR), upregulating key markers including GRP78, IRE1α, CHOP, and spliced XBP1 (XBP1s) at both mRNA and protein levels. Co-treatment with the ER stress inhibitor 4-phenylbutyrate (4-PBA) only partially reversed these effects, suggesting robust ER stress activation by RS-PP-059. In parallel, RS-PP-059 increased intracellular reactive oxygen species (ROS) in a time-dependent manner, accompanied by differential regulation of antioxidant genes with strong induction of HO-1 and suppression of CAT, SOD1, and GPX-1. Importantly, pretreatment with N-acetyl-L-cysteine (NAC) abolished ROS accumulation, ER stress activation, apoptosis, and loss of viability, confirming the ROS-dependent mechanism. In conclusion, our findings demonstrate that RS-PP-059 exerts potent anticancer effects in colorectal cancer cells by promoting ROS-mediated ER stress, leading to DNA damage and apoptosis.