Anticancer properties of Saraca Asoca (ROXB.) De Wilde and its allied species Kingiodendron Pinnatum (ROXB. Ex Dc.) harms with special emphasis on breast cancer

Abstract

Saraca asoca, commonly known as Asoka is the primary component in Asokarishta, an Ayurvedic polyherbal preparation for treating gynecological conditions in women. Various pharmacological and biological properties including antioxidant, antibacterial, anti- inflammatory, anti-keratinization and others have been reported in this plant. Kingiodendron pinnatum, known as Malabar mahogany is used as an alternative for S. asoca in Asokarishta, has shown similar phytochemical composition and efficacy. While, K. pinnatum has been shown to possess antioxidant and anti-inflammatory qualities, its other biological properties remain underexplored. Therefore, the present study sought to investigate the anticancer potential of S. asoca and K. pinnatum with a special emphasis on breast cancer. The crude methanol extracts of S. asoca and K. pinnatum bark demonstrated cytotoxic effect on murine DLA and EAC tumour cells and exhibited significant antitumour properties on mouse solid and ascites tumour models. In MTT assay, the extract shows potent antiproliferative effect on cell lines of breast cancer expressing ERβ, including MDA-MB-231, MDA-MB-468 and SK-BR-3. Similar results were observed on prostate, colorectal and cervical cancer cell lines, which also express ERβ. However, despite at high concentrations, the extracts didn’t exhibit anti-proliferative effects on the ERα expressing cell line, MCF-7. Given the low efficacy of conventional chemotherapy in treating triple- negative breast cancer, this particular cytotoxicity against ERβ expressing breast cell lines is noteworthy. Phytochemical study of both plant extracts revealed the existence of several compounds like phenols, alkaloids, saponins, flavonoids, terpenoids, phytosterols and tannins. Numerous functional groups, including aldehydes, alkenes, amines, aromatics, carboxylic acids, esters, phenols, and others, were identified by FT-IR analysis. Chromatographic techniques, such as HPTLC, HPLC, and LC-MS, disclosed the existence of phytoestrogenic compounds like β-sitosterol, quercetin, kaempferol, and others. This raises the possibility that the phytoestrogens identified in the plants may have an agonistic effect on cells expressing ERβ. Consequently molecular docking of phytoestrogens with targeted estrogen receptors were done and results shows that phytoestrogens such as quercetin and kaempferol bind to the ERβ more deeply than the ERα. The in vitro anti-inflammatory capabilities of plant extracts was demonstrated through theinhibition of 5-lipoxygenase enzyme activity and scavenging of NO radicals in LPS- activated RAW 264.7 cells. The extracts effectively decreased inflammation in acute and chronic paw edemas in mice induced by carrageenan and formalin, respectively. The antiestrogenic ability of extracts was evaluated using the estrogen screen assay revealing proliferative effect of extracts on ERα-expressing cells and modest antiproliferative effect on ERβ-expressing breast cancer cell lines. Furthermore, the in vivo rodent uterotropic assay shows suppressed growth of the endometrial lining and reduced serum estrogen levels indicating the antiestrogenic effects of extracts. The antioxidant potency of the plants was assessed through in vitro and in vivo models. The extracts effectively inhibited or scavenged free radicals such as ABTS, DPPH, superoxide and hydroxyl, in a concentration-dependent manner. Additionally, the plant extracts considerably hindered AAPH-induced lipid peroxidation and hemolysis in human erythrocytes. Significant augmentation of the endogenous antioxidant system has been seen in the in vivo sodium fluoride intoxication model, as demonstrated by increased levels of SOD, catalase, and GSH, coupled with a reduction in lipid peroxidation. Both plant extracts substantially mitigated the structural changes in the liver tissue exposed to NaF. Considerable protective effects were shown by the extracts against DMBA induced mammary carcinogenesis in vivo. The elevation in liver marker enzymes by DMBA was mitigated by the extracts, and the histology of the mammary gland exhibited fewer invasive cancer cells and reduced cell proliferation. The extracts decreased the mRNA expression of oncogenes ER- α 1, BCL2 and c-MYC, which were elevated by DMBA except for PIN1 expression. The extract also demonstrated potent inhibition of both tumour growth and metastasis in 4T1 cell induced breast cancer in mice. Histological analysis of the mammary glands exhibited a marked reduction in malignant cell proliferation, thereby effectively preventing metastasis in the group administered with the plant extract. The possible mechanism of action of S. asoca and K. pinnatum extracts on TNBC cells was analysed in vitro by assessing cell death patterns and the results point to apoptotic-mediated cell death. Fluorescent staining with EB/AO reveals structural alterations with dispersed chromatin and perforated cells, which are indications of apoptosis. In the FRET-based caspase-3 activation assay, treatment of MDA-MB-231 cells with plant extracts activatesexecutioner caspases like caspase 3, and flow cytometry-based cell cycle analysis demonstrates arrest in the G0/G1 phase, reflected by percentage of MDA-MB-231 cells in the sub-G0/G1 phase, indicating apoptosis. In conclusion, morphological and biochemical assays indicate that the cytotoxicity exerted by S. asoca and K. pinnatum extracts, particularly against TNBCs, is through an apoptotic mechanism. The results suggest that the activation of caspase 3 likely played a significant role in the anticancer ability of both plant extracts on triple-negative breast cancer cells, by inducing apoptosis. This opens a promising avenue for further research addressing TNBCs, and additional exploration is warranted to understand the influence of phytoestrogens on cancers expressing ERβ. Compounds capable of binding to ERβ have the potential to prevent or treat malignancies such as TNBCs. Consequently, the development of antiestrogenic drugs is crucial for impeding the growth of this subtype of cancer.