<i>N</i>,<i>N</i>-Dimethyl-anthranilic Acid from <i>Calvatia nipponica</i> Mushroom Fruiting Bodies Induces Apoptotic Effects on MDA-MB-231 Human Breast Cancer Cells

Breast cancer ranks among the most prevalent malignancies affecting women worldwide, and apoptosis-targeting drugs are attractive candidates for the treatment of cancer. In the current study, we investigated the in vitro cytotoxicity of the mushroom <i>Calvatia nipponica</i> in human breast cancer cells (MDA-MB-231), identified potential antitumor compounds through bioactivity-guided isolation, and elucidated the antitumor, pro-apoptotic molecular mechanisms of the identified bioactive compounds. <i>C. nipponica</i> is edible when young, and it has been used as a food source as well as a traditional medicine in wound dressings. However, only a limited number of studies have reported its chemical composition and biological activities. In the screening test, the methanol extract of <i>C. nipponica</i> fruiting bodies exhibited cytotoxicity against MDA-MB-231 cells. Bioactivity-guided fractionation of the methanol (MeOH) extract and chemical investigation of the active fractions resulted in the isolation of fourteen compounds (<b>1</b>–<b>14</b>), including six alkaloids (<b>1</b>–<b>3</b>, <b>5</b>, <b>7</b>, and <b>8</b>), two phenolic compounds (<b>4</b> and <b>6</b>), one fatty acid (<b>9</b>), and five steroids (<b>10</b>–<b>14</b>). The structures of the isolated compounds were determined using NMR spectroscopic methods, liquid chromatography–mass spectrometry, and comparison of data with previously reported values. The isolated compounds (<b>1</b>–<b>14</b>) were tested for cytotoxicity against MDA-MB-231 cells, where compound <b>1</b>, i.e., <i>N</i>,<i>N</i>-dimethyl-anthranilic acid, exhibited the most significant cytotoxicity against MDA-MB-231 cells, with an IC₅₀ value of 90.28 ± 4.23 μM and apoptotic cell death of 56.01% ± 2.64% at 100 μM. Treatment with compound <b>1</b> resulted in an upregulation of protein levels, including cleaved caspase-8, cleaved poly (ADP-ribose) polymerase, Bcl-2-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, Bad, and Cytochrome <i>c</i>, but decreased the levels of B-cell lymphoma 2 (Bcl-2). Overall, these results indicate that <i>N</i>,<i>N</i>-dimethyl-anthranilic acid (<b>1</b>) may have anti-breast cancer activity and is probably involved in the induction of apoptosis mediated by extrinsic and intrinsic signaling pathways.