Cepharanthine as a Biofactor: Molecular Functions, Signaling Pathway Modulation and Therapeutic Relevance in Cancer and Drug Resistance.

Cepharanthine(CEP), a biscoclurine alkaloid isolated from StephaniacepharanthaHayata, has demonstrated anticancer activity in several different types of cancer cells. Colorectal cancer(CRC) is one of the most common cancers in both men and women. Mutated p53 in CRC was reported to be associated with resistance to commonly used chemotherapeutic agents including, 5‑fluorouracil, oxaliplatin and irinotecan. Many studies reported that mutation of p53 induced chemoresistance through several mechanisms, including induction of drug efflux, disruption of cell cycle regulation, evasion of apoptosis and upregulation of DNA repair. This study aimed to evaluate the anticancer activity of CEP in p53 mutant versus p53 wild-type colorectal cancer cells and determine its underlying mechanisms of action. Our results showed that CEP induced colorectal cancer cell death in a concentration-dependent manner. Remarkably, CEP was more effective in controlling the growth of the p53 mutant colorectal cancer cell lines, HT‑29 and SW-620, than the p53 wild-type colorectal cancer cell lines, COLO‑205 and HCT-116. Further studies on the underlying mechanisms revealed that CEP could induce cell cycle arrest and apoptosis in both HT‑29 and COLO‑205 cells. Treatment with CEP dramatically increased p21Waf1/Cip1 expression levels of the p53 mutant cell line HT‑29 and to a lesser extent, the p53 wild-type cell line COLO‑205. In addition, cyclinA and Bcl‑2 expression levels of both cell lines were significantly downregulated following treatment with CEP. CEP also induced ROS formation in colorectal cancer cells. Taken together, we concluded that CEP effectively induced cell cycle arrest and apoptosis which may be mediated through upregulation of p21Waf1/Cip1, downregulation of cyclinA and Bcl‑2 and induction of ROS production in colorectal cancer cells. These findings suggested that CEP could potentially be a novel anticancer agent for p53 mutant colorectal cancer cells which are often resistant to current chemotherapeutic agents.

Neuroblastoma (NB) is the most common extra cranial solid tumor of childhood and accounts for approximately 15% of all cancers in children less than 5 years of age. At time of diagnosis, most children already have metastatic disease. Despite multimodality treatment for advanced NB, the survival rate is only 30%; therefore there is a clinical need for more effective therapeutic strategies. Cepharanthine (CEP), a natural alkaloid extracted from Stephania Cepharantha Hayata, has been widely used in Japan for years to treat both acute and chronic diseases without serious side effects. We examined the potential of cepharanthine to induce cell death in several NB cell lines. We exposed SMSKCNR (SMSR), SK-N-BE2c (BE2c), and SH-SY5Y (SY5Y) NB cells to increasing concentrations of CEP and examined cell viability with MTS assay. While 1μM treatment had no significant effect on cell viability at 72 hours, 10μM CEP significantly reduced cell viability compared to vehicle-treated controls (SMSR 11±2.2%; BE2c 8.2±1.1%; SY5Y 1.2±4.1%, p<0.01). In comparison 10μM CEP did not affect PC3 prostate cancer cell viability (98±2.9% of non-treated controls). Next we examined the effect of CEP in combination with vincristine (Vin), a vinca alkaloid commonly used for NB treatment. NB cells were treated with 3.0μM CEP, 50nM Vin, or both. At 72 hours, 3.0μM CEP alone reduced viability to 72±3.4% (SMSR), 83±3.7% (BE2c), and 82±7.5% (SY5Y) of vehicle-treated controls. While Vin treatment alone had no affect on viability, 92±4.3% (BE2c), 104±4.7% (SMSR), and 94±7.5% (SY5Y), the combination of Vin and CEP significantly reduced viability to less than 10% of non-treated controls (SMSR 7.2±3.8%, BE2c 8.8±1.0%, and SY5Y 2.8±7.0%, p<0.01). To determine if CEP treatment increased Vin-induced cell death by reversing multidrug resistance, we examined the effect of CEP on doxorubicin (DOX) cellular accumulation. DOX is an anthracycline commonly used in chemotherapy and possesses fluorescent characteristics. In addition, both DOX and Vin are known substrates for multidrug drug resistance proteins. Be2c NB cells were treated with 3.0μM DOX with and without 10μM CEP for one hour and the level of DOX (n = 100 nuclei) evaluated with fluorescent microscopy. Nuclear retention of DOX was evaluated 18 hours after removing DOX and CEP from the culture media. Using fluorescent microscopy, the intensity of DOX signal was approximately 3-fold higher in cells treated with CEP (CEP+DOX: 59±19.2 vs. DOX: 20±19.1 arbitrary units; p<0.01) suggesting an inhibition of multidrug resistance proteins by CEP. Our data indicates that CEP can directly induce NB cell death as well as sensitize cells to Vin-induced cell death at concentrations that are clinically achievable. CEP represents a novel addition to NB treatment regimens and may potentially improve clinical outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3553.

A variety of organisms, such as bacteria, fungi, and plants, produce secondary metabolites, also known as natural products. Natural products have been a prolific source and an inspiration for numerous medical agents with widely divergent chemical structures and biological activities, including antimicrobial, immunosuppressive, anticancer, and anti-inflammatory activities, many of which have been developed as treatments and have potential therapeutic applications for human diseases. Aside from natural products, the recent development of recombinant DNA technology has sparked the development of a wide array of biopharmaceutical products, such as recombinant proteins, offering significant advances in treating a broad spectrum of medical illnesses and conditions. Herein, we will introduce the structures and diverse biological activities of natural products and recombinant proteins that have been exploited as valuable molecules in medicine, agriculture and insect control. In addition, we will explore past and ongoing efforts along with achievements in the development of robust and promising microorganisms as cell factories to produce biologically active molecules. Furthermore, we will review multi-disciplinary and comprehensive engineering approaches directed at improving yields of microbial production of natural products and proteins and generating novel molecules. Throughout this article, we will suggest ways in which microbial-derived biologically active molecular entities and their analogs could continue to inspire the development of new therapeutic agents in academia and industry.

Inhibition of herpes simplex virus 1 by cepharanthine via promoting cellular autophagy through up-regulation of STING/TBK1/P62 pathway

The development of new anticancer therapies remains challenging due to tumor heterogeneity and the frequent emergence of multidrug resistance (MDR). Natural products have garnered increasing attention as alternative or complementary therapeutic agents due to their bioactivity and reduced toxicity. Polyphenols, particularly curcumin and its derivatives, have shown promise in modulating signaling pathways, enhancing chemosensitivity, and overcoming drug resistance. The anticancer potential of dimethoxycurcumin, a chemically modified curcumin derivative identified through consensus fingerprint similarity screening, was investigated for its potential to inhibit ABCC3 (MRP3)-a member of the ATP-binding cassette (ABC) transporter family implicated in drug efflux, tumor cell survival, and resistance. In vitro experiments demonstrated that dimethoxycurcumin significantly reduced cancer cell viability and colony formation, indicating a strong inhibitory effect on ABCC3 function. These results suggest that dimethoxycurcumin may sensitize cancer cells to chemotherapy by targeting resistance pathways. The data presented contribute to the growing body of evidence suggesting that bioactive plant-derived compounds, including chemically modified derivatives, may hold therapeutic potential in oncology by modulating multidrug resistance pathways. Targeting ABC transporters with natural compound derivatives could offer a promising strategy for developing more effective and less toxic anticancer therapies.

Pyridines have occupied a unique place in medicinal chemistry as it is widely profound as natural products and formed the integral backbone of great number of drugs in the market. In particular, 3-cyano-2-oxa-pyridines showed diverse biological and pharmacological activities such as cardiotonic, antimicrobial, antidepressant, and anticancer activity. 3-Cyano-2-oxa-pyridine derivatives have elevated importance for modern medicinal applications especially in cancer therapy. This article shed light on the general chemical synthetic approaches of 3-cyano-2-oxa-pyridines and summarized their various biological activities and pharmacological uses. This article may be helpful in the future to direct attention towards utilization of 3-cyano-2-oxa-pyridine template in the design of new molecules with enhanced biological properties such as PIM1 kinase, tubulin polymerase and survivin inhibitors for cancer therapy or new AMPK activator for diabetes and obesity control or cardiotonic agents.

Cancer is one of the leading causes of death worldwide and despite significant improvements to treatment and prevention, cancer cases remain on the rise. Chemotherapy is used to treat patients with cancer, however these do not only kill the cancer cells but also kill normal, healthy cells in the patient. Furthermore, cancer cells have the capacity to become resistant to chemotherapeutic treatment. Therefore, new treatments need to be developed to overcome this problem. One avenue that is being researched is the use of natural products as chemotherapeutic drugs. Over 60% of anticancer drugs used today are either natural products, or their synthetic derivatives and new research is being performed to screen plant and animal secondary metabolites to discover new compounds with anti-cancer therapeutic potential. The research reported in this thesis uses two compounds purified from natural products to explore a novel approach for cancer treatment. Stress granules (SGs) are messenger ribonucleoprotein particles that are produced in the cytoplasm of the cell in response to stress. Stress granules have been linked to the inhibition of apoptosis and development of multiple drug resistance and it has been suggested that cancer cells can hijack stress granules and use their biological activities to enhance cancer cell survival. In a study by Fournier et al the inhibition of stress granules in bortezomib resistant cancer cells allowed these cells to become sensitive to bortezomib treatment and resulted in an increase in cell death from 15% to 75% (Fournier et al., 2010). This suggests that the inhibition of stress granule formation may restore chemo-sensitivity to the cancer cells, however, the full effect has not been explored beyond the cell based experiments described by Fournier et al. This research project was based on the research by Fournier et al, suggesting that SG inhibition can increase the efficacy of bortezomib. The aims of this project were to discover natural products that inhibited SG formation and use these natural products in combination with the chemotherapeutics bortezomib and sorafenib to increase their efficacies. Chapter 3 describes the optimisation and characterisation of SG formation in HEK293, MCF7, T47D, Vero, HeLa, MDAMB231 and MCF7MDR cells for the development of a SG inhibition assay. Chapter 4 describes the screening of 36 compounds from the Davis Open Access Compound Library from which 2 compounds, RAD112 and psammaplysin F were discovered; having activity inhibiting SG formation in the in vitro SG inhibition assay. In chapter 5, the mechanism of action of RAD112 and psammaplysin F were explored. RAD112 belongs to the chalcone class and this class of compound is known for the disruption of microtubules. A microtubule assay was performed analysing the effect of RAD112 against a known microtubule inhibitor, nocodazole and it was confirmed that RAD112 was disrupting microtubules. Psammaplysin F did not cause the disruption of microtubules, therefore, the most common pathway of SG formation, phosphorylation of eIF2α (p-eIF2α) was analysed. It was discovered that psammaplysin F was reducing the amount of p-eIF2α in HEK293, MCF7, Vero and MCF7MDR cells. The mechanism of action studies of both compounds show promising results that warrant further evaluation. Combinational therapies have many advantages over single chemotherapy regimes in breast cancer as it has been shown that it can increase the patients disease free survival rate and reduce the risk of reoccurrence. In chapter 6 RAD112 and psammaplysin F were used in combination with bortezomib or sorafenib and the interaction between RAD112 with bortezomib or sorafenib and psammaplysin F with bortezomib or sorafenib was determined by cell viability assays and analysed using Compusyn software. The increase in efficacy of bortezomib or sorafenib when combined with RAD112 and psammaplysin F was also examined in the in vitro combinational assay. All combinations of the compounds with the drugs resulted in a synergistic interaction in most cell lines. However, psammaplysin F and sorafenib had the strongest synergistic interaction in MCF7MDR cells, with a combination index (CI) value of <0.4. The IC50 of sorafenib was decreased 4 fold in MCF7MDR cells and 7 fold in MCF7 cells when combined with psammaplysin F. The efficacy of bortezomib and sorafenib was increased after treatment with RAD112 and psammaplysin F suggesting that psammaplysin F and bortezomib have the potential to be used in combination with known chemotherapeutics to restore drug efficacy. Altogether, this thesis has identified two compounds that inhibit SG formation, RAD112 and psammaplysin F. The mechanism of action studies has revealed two different mechanisms of action for SG inhibition, microtubule inhibition and inhibition of p-eIF2α for RAD112 and psammaplysin F respectively. Combinational studies has resulted in synergistic interactions between RAD112 and bortezomib or sorafenib and psammaplysin F and bortezomib and sorafenib and increased efficacy of bortezomib and sorafenib. These findings show promise as a new strategy in the battle against cancer and further studies involving the complete mechanism of action of these compounds have to be carried out before they can move onto pre-clinical evaluation.

The expression of HNF1 homeobox B (HNF1B) is associated with cancer risk in several tumors, including ovarian cancer, and its decreased expression play roles in cancer development. However, the study of HNF1B and cancer is limited, and its association with drug resistance in cancer has never been reported. On the basis of array data retrieved from Oncomine and Gene Expression Omnibus(GEO) online database, we found that the mRNA expression of HNF1B in 586ovarian serous cystadenocarcinomas and in platinum-resistant A2780 epithelial ovarian cancer cells was significantly decreased, indicating a potential role of HNF1B in drug resistance in ovarian cancer. Based on this finding, comprehensive bioinformatics analyses, including protein/gene interaction, protein-small molecule/chemical interaction, biological process annotation, gene co-occurrence and pathway enrichment analysis and microRNA-mRNA interaction, were performed to illustrate the association of HNF1B with drug resistance in ovarian cancer. We found that among the proteins/genes, small molecules/chemicals and microRNAs which directly interacted with HNF1B, the majority was associated with drug resistance in cancer, particularly in ovarian cancer. Biological process annotation revealed that HNF1B closely related to 24 biological processes which were all notably associated with ovarian cancer and drug resistance. These results indicated that the downregulation of HNF1B may contribute to drug resistance in ovarian cancer, via its direct interactions with these drug resistance-related proteins/genes, small molecules/chemicals and microRNAs, and via its regulations on the drug resistance-related biological processes. Pathway enrichment analysis of 36 genes which co-occurred with HNF1B, ovarian cancer and drug resistance indicated that the HNF1B may perform its drug resistance-related functions through 4 pathways including ErbB signaling, focal adhesion, apoptosis and p53 signaling. Collectively, in this study, we illustrated for the first time that HNF1B may contribute to drug resistance in ovarian cancer, potentially through the 4 pathways. The present study may pave the way for further investigation of the drug resistance-related functions of HNF1B in ovarian cancer.

Editorial: Signalling pathways in anti-cancer drug resistance.

Glioblastoma multiforme (GBM) comprises the largest group of brain tumors which are drug resistant and respond very poorly to the current therapies. In this study, we used sulforaphane (SFN), a multi-targeting agent with cancer preventive and anti-cancer activities and showed that it targets GBM established cell lines, early primary cultures, and CD133+ GBM stem cells as well as in GBM stem-like spheroids. SFN at 5-50 μM triggered significant inhibition of cell survival and induced apoptotic cell death in GBM cells and CD133+ stem cells isolated from four GBM cell lines. SFN induced apoptosis in U87MG cells was associated with caspase-7 activation. Moreover, SFN triggered formation of intracellular reactive oxygen species (ROS) and when the cells were pre-treated with 10 mM N-acetyl cysteine (NAC), ROS production and cell survival in cells treated with 5-10 μM were similar to the control untreated U87MG cells, revealing that SFN-triggered cell death is ROS-dependent. Moreover, SFN-generated ROS in U87MG cells were formed at the Mitochondrial Respiratory Chain (MRC) level. SFN also increased expression of the TRAIL receptor DR5 in GBM cells, U87MG and SF767 cells by 24 h post-exposure. Moreover, as revealed by comet assay, SFN increased single- and double-strand DNA breaks in GBM. Compared to untreated control cells, a significantly higher amount of γ-H2AX foci and as consequence higher number of DNA double-strand breaks (DSBs) breaks were observed in the SFN-treated sample. In vivo studies, using NOD/SCID mice revealed that SFN administration via oral gavage at 100 mg/kg for 3 cycles significantly decreases the growth of ectopic xenografts established from the early passage primary cultures of GBM10. Our results show that SFN robustly inhibits growth of GBM cells in vitro and in vivo and induces cell death in established cell cultures, early passage primary cultures, as well as it is effective in eliminating GBM cancer stem cells, which play a major role in drug resistance and disease recurrence. These results suggest that use of SFN alone or in combination with other agents, may potentially improve survival of brain tumor patients. Citation Format: Khadijeh Bijangi-Vishehsaraei, Mohammad R. Saadatzadeh, Haiyan Wang, Malgorzata M. Kamocka, Wenjing Cai, Aaron A. Cohen-Gadol, Stacey L. Halum, Karen E. Pollok, Jann N. Sarkaria, Ahmad R. Safa. Sulforaphane depresses proliferation and induces cell death in glioblastoma multiforme (GBM) cells, GBM stem cell-like spheroids, and tumor xenografts through modulation of multiple cell signaling pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2267. doi:10.1158/1538-7445.AM2014-2267

Cancer remains a significant global health concern, with millions of new cases and deaths recorded annually. The limitations of conventional therapies, including chemotherapy and radiation, have driven the search for potent and less toxic alternatives. Natural products have emerged as promising candidates for cancer treatment due to their diverse biological activities, such as antioxidant, anti-inflammatory, and immunomodulatory effects. This review explores the anticancer potential of various natural products derived from plants, marine organisms, and microorganisms. These natural compounds, including flavonoids, alkaloids, terpenoids, polyphenols, and others, have demonstrated multiple mechanisms of action, such as inducing apoptosis, inhibiting cancer cell proliferation, and modulating signalling pathways. Despite the challenges in development, such as bioavailability, regulatory hurdles, and intellectual property issues, natural products continue to offer valuable insights and opportunities for innovative cancer therapies. The review highlights the importance of integrating natural products into modern therapeutic regimens to enhance the efficacy and safety of cancer treatments, emphasizing the need for further research to realize their full potential.

Dietary polyphenols suppress chronic inflammation by modulation of multiple inflammation-associated cell signaling pathways

Osteogenic differentiation of valve interstitial cells (VICs) directly leads to aortic valve calcification, which is a common cardiovascular disease caused by inflammation and metabolic disorder. There is still no ideal drug for its treatment and prevention. The purpose of this study was to explore the effect and molecular mechanism of cepharanthine (CEP), a natural product, on inhibiting the osteogenic differentiation of VICs. First, CCK8 assay was used to evaluate cell viability of CEP on VICs. CEP concentration of 10 μM was the effective dose with slight cytotoxicity, which was used for further study. The alizarin red staining analysis showed that CEP significantly inhibited calcium deposition caused by osteogenic medium related calcification induction. In order to explore the anti-calcification molecular mechanism of CEP, transcriptome and metabolome were synchronously used to discover the possible molecular mechanism and target of CEP. The results showed that CEP inhibited valve calcification by regulating the glycolytic pathway. The molecular docking of CEP and selected key factors in glycolysis showed significant binding energies for GLUT1 (−11.3 kcal/mol), ENO1 (−10.6 kcal/mol), PKM (−9.8 kcal/mol), HK2 (−9.2 kcal/mol), PFKM (−9.0 kcal/mol), and PFKP (−8.9 kcal/mol). The correlation analysis of RUNX2 expression and cellular lactate content showed R2 of 0.7 (p < 0.001). In conclusion, this study demonstrated that CEP inhibited osteoblastic differentiation of VICs by interfering with glycolytic metabolisms via downregulation of the production of lactate and glycolysis-associated metabolites.

Resistance to doxorubicin (DOX) is mainly due to the effect of P-glycoprotein encoded by the multidrug resistance (MDR) gene. Cepharanthine (CEP) has been shown to circumvent multidrug resistance in P-glycoprotein-expressing cell lines. In the present study, we investigated the augmentation of DOX sensitivity by CEP using an MTT assay, and assessed the correlation between DOX sensitivity and P-glycoprotein expression by flow cytometry, in highly purified fresh human tumor cells obtained from 73 cancer patients. DOX sensitivity was decreased in proportion to P-glycoprotein expression. The cytotoxicity of DOX was increased by CEP in tumor cells possessing low DOX sensitivity. Moreover, there was a significant correlation between the effect of CEP on cytotoxicity and P-glycoprotein expression. Thus, CEP might be able to circumvent DOX resistance in cancer patients.

Exploring the therapeutic potential of benzodioxane carboxylic acid-based hydrazones: Structural, computational, and biological insights