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Context: Piper cubeba L. (family Piperaceae) is used traditionally for the treatment of many diseases, including cancer. Aims: To determine the anticancer activity of P. cubeba crude extracts on cancer cells and identify chemical constituents of the active fraction. Methods: Seeds of P. cubeba were sequentially extracted with dichloromethane followed by methanol and purified using column chromatography. Fractions were screened the cytotoxicity against triple negative breast cancer (MDA-MB-468) using MTT assay. Then, active fractions were evaluated cytotoxicity against breast cancer (MCF-7 and MDA-MB-231), colon cancer (HT-29), cholangiocarcinoma (KKU-M213) and normal fibroblast (L929) cells. Total phenolic, tannin, and flavonoid contents of active fraction were investigated, and the chemical composition was analyzed using GC-MS. Flow cytometry was applied to determine the cell cycle, apoptosis, and evidence of caspase activation. Results: Fraction DE14 and DE15 showed cytotoxic activity against MDA-MB-468. Fraction DE15 exhibited the most potent cytotoxicity against MDA-MB-231, KKU-M213, HT-29 and L929. As the fraction DE15 was of dichloromethane extraction, major contents were nonpolar fatty acids and fatty acid esters, followed by propylene glycol and hydrocarbons. The phenolic compounds were however traceable, leading to their measurable antioxidant property. This extract did not cause cell cycle arrest on MDA-MB-231. However, fraction DE15 significantly increased apoptotic cells at 48 and 72 h, and significantly induced multi-caspases activity on MDA-MB-231 in a time-dependent manner. Conclusions: Fraction DE15 exhibited cytotoxicity effect and induced apoptosis on triple negative breast cancer and showed less toxicity on normal fibroblast cells.

Trans-(−)-kusunokinin, an anticancer compound, binds CSF1R with low affinity in breast cancer cells. Therefore, finding an additional possible target of trans-(−)-kusunokinin remains of importance for further development. Here, a computational study was completed followed by indirect proof of specific target proteins using small interfering RNA (siRNA). Ten proteins in breast cancer were selected for molecular docking and molecular dynamics simulation. A preferred active form in racemic trans-(±)-kusunokinin was trans-(−)-kusunokinin, which had stronger binding energy on HER2 trans-(+)-kusunokinin; however, it was weaker than the designed HER inhibitors (03Q and neratinib). Predictively, trans-(−)-kusunokinin bound HER2 similarly to a reversible HER2 inhibitor. We then verified the action of (±)-kusunokinin compared with neratinibon breast cancer cells (MCF-7). (±)-Kusunokinin exhibited less cytotoxicity on normal L-929 and MCF-7 than neratinib. (±)-Kusunokinin and neratinib had stronger inhibited cell proliferation than siRNA-HER2. Moreover, (±)-kusunokinin decreased Ras, ERK, CyclinB1, CyclinD and CDK1. Meanwhile, neratinib downregulated HER, MEK1, ERK, c-Myc, CyclinB1, CyclinD and CDK1. Knocking down HER2 downregulated only HER2. siRNA-HER2 combination with (±)-kusunokinin suppressed HER2, c-Myc, CyclinB1, CyclinD and CDK1. On the other hand, siRNA-HER2 combination with neratinib increased HER2, MEK1, ERK, c-Myc, CyclinB1, CyclinD and CDK1 to normal levels. We conclude that trans-(±)-kusunokinin may bind HER2 with low affinity and had a different action from neratinib.

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC 50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.

Synthetic trans-()-kusunokinin (()KU), a potential anticancer substance, was revealed to have an inhibitory effect on breast cancer. According to the computational modeling prediction, AKR1B1, an oxidative stress and cancer migration protein, could be a target protein of trans-()-kusunokinin. In this study, we determined the binding of ()KU and AKR1B1 on triple-negative breast and non-serous ovarian cancers. We found that ()KU exhibited a cytotoxic effect that was significantly stronger than zopolrestat (ZP) and epalrestat (EP) (known AKR1B1 inhibitors) on breast and ovarian cancer cells. ()KU inhibited aldose reductase activity that was stronger than trans-()-arctiin (()AR) but weaker than ZP and EP. Interestingly, ()KU stabilized AKR1B1 on SKOV3 and Hs578T cells after being heated at 60 and 75 °C, respectively. ()KU decreased malondialdehyde (MDA), an oxidative stress marker, on Hs578T cells in a dose-dependent manner and the suppression was stronger than EP. Furthermore, ()KU downregulated AKR1B1 and its downstream proteins, including PKC-δ, NF-κB, AKT, Nrf2, COX2, Twist2 and N-cadherin and up-regulated E-cadherin. ()KU showed an inhibitory effect on AKR1B1 and its downstream proteins, similar to siRNA–AKR1B1. Interestingly, the combination of siRNA–AKR1B1 with EP or ()KU showed a greater effect on the suppression of AKR1B1, N-cadherin, E-cadherin and NF-κB than single treatments. Taken together, we concluded that ()KU-bound AKR1B1 leads to the attenuation of cellular oxidative stress, as well as the aggressiveness of breast cancer cell migration.