Article pubs.acs.org/jnp
6‑Acetonyldihydrochelerythrine Is a Potent Inducer of Apoptosis in HCT116 and SW620 Colon Cancer Cells Tayyab A. Mansoor,†,‡ Pedro M. Borralho,†,‡ Xuan Luo,† Silva Mulhovo,§ Cecília M. P. Rodrigues,† and Maria-José U. Ferreira*,† †
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, University of Lisbon, 1649-003 Lisbon, Portugal Centro de Estudos Moçambicanos e de Etnociências, Faculty of Natural Sciences and Mathematics, Pedagogical University, 21402161 Maputo, Mozambique
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ABSTRACT: 6-Acetonyldihydrochelerythrine (1), a benzophenanthridine alkaloid, isolated from the methanol extract of Zanthoxylum capense, displayed potent cytotoxic activity in human HCT116 and SW620 colon carcinoma cells, to a higher extent than 5-fluorouracil (5-FU), the cornerstone chemotherapeutic agent in colon cancer. Cytotoxicity of 1 was evaluated by MTS, lactate dehydrogenase (LDH), and Guava ViaCount assays. Interestingly, 1 significantly induced cytotoxicity in both cell lines, leading to a significant increase in LDH release, as compared to 5-FU. Further, Guava ViaCount flow cytometry assays demonstrated that 1 significantly increased cell death, as shown by the presence of a significantly higher population of apoptotic cells in both cell lines, as compared to cells exposed to 5-FU. Furthermore, evaluation of nuclear morphology by Hoechst staining of 1-treated HCT116 and SW620 cells confirmed flow cytometry results, demonstrating a marked induction of apoptotic cell death by 1, again to a further extent than that elicited by 5-FU. In addition, immunoblot analysis to ascertain the molecular events triggered by 1 exposure was performed. The results show that 1 exposure reduced the steady-state expression and activation of the pro-survival proteins ERK5 and Akt and increased the steady-state expression of p53 in both HCT116 and SW620 cells. Changes in ERK5 or Akt activation can be ascertained by evaluating the ratio of p-ERK5/ERK5 or p-Akt/Akt. In addition, exposure to 1 reduced expression of XIAP, Bcl-XL, and Bcl-2, while increasing the cleavage of poly(ADP-ribose) polymerase in both cell lines. Collectively, the data indicate that 6-acetonyldihydrochelerythrine (1) is a potent inducer of apoptosis in HCT116 and SW620 cell lines, highlighting its potential relevance in colon cancer.
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tion, chromatin condensation, and chromosomal DNA fragmentation, finally leading to the death of the cell.3 The discovery of new therapeutic agents that can induce apoptosis in cancer cells and the elucidation of the pathways by which they operate are of prime interest in anticancer drug development. Importantly, colorectal cancer (CRC) is a cancer type of major relevance, particularly in the developed world, displaying high incidence and associated mortality.4 Thus far, synthetic chemical compounds, such as 5-fluorouracil (5-FU), are still the first choice of treatment, especially for CRC patients in advanced stages, together with specific biological agents. However, 5-FU resistance is one of the leading obstacles in CRC successful chemotherapy, as this agent remains the cornerstone agent in the treatment of CRC.5 Medicinal plants have long been recognized as a relevant source of medicines.6 In fact, the majority of anticancer agents currently available in the market are either directly isolated
uring chemotherapy, when cancer cells succeed in evading anticancer agent effects, the end result is unsuccessful cancer treatment. Many anticancer agents have consumed decades of research and development efforts to reach the clinic; however, after some time, these drugs lose their efficacy due to the development of resistance. 1 The simultaneous resistance of cancer cells to many functionally and structurally unrelated anticancer drugs is known as multidrug resistance (MDR), a multifactorial phenomenon that can result from several mechanisms including a failure to undergo apoptosis. Most chemotherapeutic agents exert their anticancer activity by inducing apoptosis. Therefore, deregulation of the apoptotic pathway can confer drug resistance and be a major limiting factor in the effectiveness of anticancer therapy. Several strategies are being employed to address the issue of anticancer drug resistance. One promising approach is the development of effective apoptosis inducers.2 Apoptosis is a process of programmed cell death, where controlled enzymatic chains of events lead to characteristic morphological cell changes, and cell demise. These changes include membrane blebbing, cell shrinkage, nuclear fragmenta© 2014 American Chemical Society and American Society of Pharmacognosy
Received: March 11, 2014 Published: July 28, 2014 1825
dx.doi.org/10.1021/np500161n | J. Nat. Prod. 2014, 77, 1825−1830
Journal of Natural Products
Article
IC50 concentration did not induce LDH release in HCT116 and resulted in only a modest 1.1-fold increase in SW620 cells. In parallel, 1 exposure at 2-fold IC50 was the most toxic, increasing cell death up to 1.9-fold in HCT116 cells and 1.6fold in SW620 cells, compared to vehicle (DMSO) control (p < 0.01). This is in contrast with 5-FU exposure, which did not lead to increased LDH release in HCT116 and again only induced a modest 1.1-fold increase in SW620 cells compared to vehicle (DMSO) control (p < 0.05) (Figure 1B). Subsequently, we performed flow cytometric analysis using the Guava ViaCount assay, to determine the percentage of viable, mid-apoptotic, and dead cells following exposure to 1 and 5-FU. Our results demonstrated that 1 significantly induced cell death following 72 h exposure to IC50 and 2-fold IC50 concentrations, in both cancer cell lines (p < 0.01) (Figure 2). Importantly, 1 at IC50 concentration was able to significantly increase the incidence of apoptosis up to 25% and 34% in HCT116 and SW620 cells, respectively, as compared to vehicle (DMSO) control exposure (p < 0.01) (Figure 2). Further, at 2fold IC50 concentration, compound 1 increased apoptotic cells up to ∼65% in HCT116 and SW620 cells (p < 0.01) (Figure 2). In contrast, 5-FU exposure at IC50 and 2 × IC 50 concentration induced significantly less apoptotic cell death as compared to 1 (p < 0.01), leading to less than15% midapoptotic cells in HCT116 and SW620 cells. Collectively, these results demonstrate a higher apoptosis induction by 1 compared to 5-FU, in both HCT116 and SW620 colon cancer cell lines. To confirm apoptosis induction by 1 exposure, nuclear morphological evaluation studies were performed by fluorescent microscopy following Hoechst’s staining. Apoptosis is characterized by distinctive morphologic as well as biochemical changes, including cell shrinkage, loss of intercellular membrane contact, progressive condensation of chromatin and cytoplasm, and subsequent nuclear fragmentation. These events culminate in the characteristic formation of apoptotic bodies, consisting of nuclear fragments and intact cell organelles surrounded by a plasma membrane. HCT116 and SW620 cells, incubated with 1 and 5-FU at IC50 and 2-fold IC50 compound concentration, and changes in nuclear morphology were evaluated at 24 h of compound exposure. The data confirmed that 1 induced higher levels of apoptosis at IC50 concentration, leading to 19% and 22% apoptotic cells in HCT116 and SW620 cells, respectively, compared to vehicle under (DMSO) control cells, which displayed less than 5% apoptotic cells (p < 0.01). Further, 1 exposure at 2-fold IC50 concentration led to 22% and 27% apoptotic cells in HCT116 and SW620 cells, respectively, as compared to vehicle (DMSO) control cells (p < 0.01) (Figure 3). The apoptosis elicited by 1 was significantly higher than that induced by the known apoptosis-inducing agent 5-FU, whose exposure at IC50 and 2-fold IC50 respectively led to
