(B) The lone and/or combined effects of oleanolic acid (OA) or LY294002 on the expression of proteins involved in MAPK kinase signaling in DU145, MCF-7, and U87 cells
(B) The lone and/or combined effects of oleanolic acid (OA) or LY294002 on the expression of proteins involved in MAPK kinase signaling in DU145, MCF-7, and U87 cells. and CDK2; and OA-treated U87 cells also exhibited G1 phase arrest caused by the increase in p-ERK, p-JNK, p-AKT, p21, and p27, and the decrease in cyclin D1, CDK4, cyclin E and CDK2. Thus, OA arrested the cell cycle at different phases and induced apoptosis in Febrifugin cancer cells. These results suggested that OA possibly altered the expression of the cell cycle regulatory proteins differently in varying types of cancer. . Thus, utmost attention is being given to searching for better and safer drugs of natural origin, which may potentially increase the efficacy of anticancer treatments . Apoptosis, or programmed cell death, is the most common mechanism used to induce cancer cell death via targeted chemotherapy . It is a regulated process that is activated by stressors such as DNA Febrifugin damage, cytokines, and oxidative stress . The p53 tumor suppressor is activated by the oncogene- or DNA damage-induced signaling pathways, which in turn accelerates the transcription of several genes involved in apoptosis such as the proapoptotic members of the Bcl-2 family, including those encoding for death receptors . Bax is an important proapoptotic member of the Bcl-2 Febrifugin family of proteins that regulates the balance between cell survival and death . In response to apoptotic signals, Bax is transformed into a fatal mitochondrial oligomer that causes mitochondrial damage, representing an important step for the intrinsic apoptotic pathway [10, 11]. Additionally, p53-induced apoptosis also activates caspases , primarily occurring through the activation of the death receptor pathway or through mitochondrial membrane depolarization . The relationship between the cell cycle and apoptosis is underscored by the role of the p53 tumor suppressor gene and those of the p21WAF1/CIP1 and genes, which induce cell cycle arrest and cell death . Cell proliferation is mediated by several signaling molecules and checkpoints that regulate cell division . The progression through the cell cycle is positively regulated by cyclin E and the cyclin-dependent kinase (CDK) complex, which phosphorylate the retinoblastoma tumor suppressor protein to induce the transition from the G1 to the S phase . However, the p21WAF1/CIP1 and p27KIP1 kinase inhibitor proteins bind to the cyclin E/CDK2 complex and block the G1/S transition . I Another protein, cyclin B1, also plays a key role in the cell cycle transition from the G2 to M phase , and the decrease in its expression levels has been suggested to disrupt cell growth and promote malignant transcription . Oleanolic acid (3–hydroxy-olea-12-en-28-oic acid; OA) is a naturally occurring pentacyclic triterpenoic acid [17, 18] that exhibits chemopreventive, hepatoprotective, tumor-suppressive, contraceptive, anti-inflammatory, antioxidant, antimicrobial, antiparasitic, antiviral, and antineoplastic characteristics [19C23]. The tumor-suppressive activity of OA was demonstrated in several cancer cell lines such as KB, HT29, MCF-7, MDA-MA-231, HCT-116, HONE-1, Hep-G2, and HL-60 [20, 24C26]. Recently, several reports showed that OA also induced G1 cell cycle arrest in the GBC-SD, NOZ, HCT15, and K562 cell lines [21, 27]. Moreover, it was reported that OA induced a concentration-dependent S phase and G2/M phase cell cycle arrest in Panc-28 and Hep-G2 cells [28, 29]. The inhibitory effects of OA were attributed to Vegfa the suppression of specific intracellular signaling pathways such as the STAT3, JNK, AKT, and NF-kappaB . As a result, these studies proposed OA as an adjunct to cancer chemotherapy. In this study, we investigated the cellular viability, apoptotic process, and cell cycle in OA-treated DU145 (prostate cancer), MCF-7 (breast cancer), and U87 (human glioblastoma) cells. Also, DU145 cell xenografts grown in BALB/C mice were injected with OA. We explored the protein expression of apoptosis, cell cycle and kinase signaling in DU145 cell xenografts grown in mice treated OA. Additionally, we also examined protein expression with respect to apoptosis, cell cycle, and kinase signaling in these OA-treated cells. The results suggested that OA differentially altered the expression of cell cycle regulatory proteins depending on the type of cancer cells. RESULTS Cytotoxic activity of OA in cancer cells To investigate the effects of OA on cell viability in cancer cell lines (DU145, MCF-7, U87), the cells were treated with 0, 25, 50, 100, and 250 g/mL OA for 24 h and cell proliferation was assessed using the MTT assay (Table ?(Table1).1). Normal, non-cancerous cell lines such as BNL Febrifugin CL. 2 (murine liver cells) and Hs 68 (human foreskin.