Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents) - Volume 19, Issue 3, 2019

Background: Head and neck squamous cell carcinoma (HNSCC) is the most common malignant cancer occurring in the head and neck area, approximately 90% of the cases. Even in the cases of primary radical treatment (surgical, concomitant chemoradiotherapy or radiotherapy alone), subsequent local recurrence or distant metastases are often observed. In patients with recurrent disease who are unable to receive radical treatment, the results of palliative chemotherapy are not satisfactory. In this review, we summarized the standard treatment options, current development of new drugs and future perspectives in the treatment of patients with recurrent locally advanced and/or metastatic HNSCC. Methods: PubMed databases with words ‘head and neck cancer treatment’, ‘immunotherapy in head and neck cancer treatment’ were searched and yielded 186512 and 2249 papers respectively. We selected the most cited articles and reports presenting new immunotherapy agents and drug combinations in HNSCC. Results: Recently, two new agents been approved in the treatment of recurrent locally advanced and/or metastatic HNSCC. These are immune-checkpoint inhibitors targeting PD1 (nivolumab and pembrolizumab) which are the most active drugs in the second line treatment of advanced HNSCC. Still, the first line ‘golden standard’ is the chemotherapy regimen (cisplatin, 5-fluorouracyl) combined with cetuximab. Many phase 3 studies are currently ongoing, evaluating the efficacy of combinational treatment-anti-CTLA4 with anti-PD1 or anti-PDL1. Very encouraging results have been shown in early phase studies evaluating the combination of immunecheckpoint inhibitors with tumor microenvironment immunosuppressive inhibitors. Conclusion: Despite the huge progress in the systemic treatment of patients with recurrent locally advanced and/or metastatic HNSCC, the disease at this stage remains incurable. Undoubtedly, further research in the field of biomarkers for effective immunotherapy is needed in order to select a group of patients whose will benefit from this therapy, as the treatment is still ineffective in most patients.

Background: MicroRNAs are noncoding RNAs which play critical roles in response to anti-cancer agents. Let-7a and miR-21 are well-known tumor-suppressor and oncomiR miRNAs, respectively. They are involved in tumorigenesis of gastric cancer and have potential to be used as markers in response to the therapy. Objective: We aimed to study alterations in the expression of Let-7a and miR-21, and their targets in gastric cancer cell lines after treatment with docetaxel. Methods: In order to determine the IC50 of docetaxel, MTT assay was performed in AGS, MKN45 and KATO III gastric cancer cell lines. The expression levels of Let-7a and miR-21 and their target genes, HMGA2 and PDCD4, were determined by reverse-transcription quantitative real-time PCR for both treated and untreated cell lines. Results: MTT assay showed higher IC50 concentration of docetaxel in KATO III in comparison with AGS and MKN45, indicating KATO III`s higher resistance to docetaxel. Following the treatment, the expression level of Let-7a was significantly increased in AGS and MKN45, while decreased in KATO III. Expression level of miR- 21 in the three treated cell lines was increased significantly. Not only Let-7a, but also expression level of HMGA2 and PDCD4 genes showed different patterns in KATO III in comparison with AGS and MKN45. Conclusion: Down-regulation and up-regulation of Let-7a in docetaxel-resistant and sensitive cell lines, respectively indicates its potential usefulness as biomarker for responsiveness of gastric cancer to the therapy with docetaxel and also for predicting patient`s outcome.

Background: Two series of 3,4-dihydropyrimidin-2(1H)-one derivatives were designed based on the main structural features characterizing reported anticancer compounds with potent VEGFR-2 inhibiting activity. Methods: All the target compounds were synthesized and investigated for their in vitro anticancer activity using MTT assay and NCI protocol. The most active compounds were further investigated for the VEGFR-2 inhibiting activity using enzyme inhibition assay. Result: Of these derivatives, compound 8b possessed significant activity against Caco-2 (IC50 of 24.9 μM) and MCF7 (IC50 of 29.4 μM), compound 10 showed excellent potency against HCT-116 (IC50 of 32.6 μM), HEPG2 (IC50 of 16.4 μM) and MCF7 (IC50 of 32.8 μM), while compound 11b exhibited moderate anticancer activity towards MCF7 (IC50 of 41.7μM). Both 8b and 10 exhibited good potency regarding the inhibition of vascular endothelial growth factor receptor 2 (VEGFR-2), with an IC50 of 14.00 and 21.62 nM, respectively. Conclusion: The activity was rationalized based on molecular docking study that supported their VEGFR-2 inhibitory activity; as indicated by their favorable binding with the active site.

Background/Objective: Forkhead Box M1 (FOXM1) is frequently activated in tumors. We studied the expression and the possible mechanism of FOXM1 and evaluated the effects of thiostrepton in an endometriotic rat model. Methods and Material: This was a randomized study in a rat model of endometriosis. Fifty female Wistar rats were surgically induced with endometriosis. After 4 weeks of observation, twenty and thirty rats were randomly allocated to an ovariectomized (OVX) group and a treatment group, respectively. The OVX group was ovariectomized and randomly divided into an OVX-estrogen group and a control (OVX -oil) group. All rats were allowed a resting period of 3 days prior to any operation. The rats in the estrogen group were given estradiol (20 μg/kg, 0.1 ml /d), while the control group was treated with an equivalent amount of sesame oil. Every group was injected with subcutaneous injection for 7 days. The treatment group was randomly divided into three groups to receive the following: TST at 150 mg/kg, ip.; TST at 250 mg/kg, ip.; or sterile normal saline, ip. The groups received these dosages every 2 days for 2 weeks. Lesion growth, histological examination, and protein expression were subsequently analyzed using caliper measurement, histology, immunostaining, and Western blot after each rat received an injection in its own group. Results: Our results showed that FOXM1 is enriched in nucleus of an ectopic endometrium when compared with a eutopic uterus. Furthermore, we found that an ERK/FOXM1/matrix metalloproteinase-9 (MMP9) signaling pathway might result in the establishment and development of endometriosis. Finally, a thiostrepton concentration dependently reduced the expression of FOXM1, MMP9 and Bcl-2 in endometriotic lesions of the treated rats. Statistical significance was accepted for a value of P < 0.05. Conclusion: We postulate that thiostrepton could inhibit the endometriotic lesions, at least in part, by decreasing the FOXM1 expression and exerting a pro-apoptotic effect. We reported for the first time that FOXM1 expresses in experimental endometriosis rat and thiostrepton may also be suitable for the administration of endometriosis by inhibiting the growth of endometriotic implants. More studies are needed to further evaluate thiostrepton’s effect.

Background: Omega-3 polyunsaturated fatty acids (omega-3 PUFAs) have significant multiple antitumor roles. However, whether epigenetic DNA hydroxymethylation enrolls in the anticancer process of omega- 3 PUFAs is still not clear yet. Objective: To expound the interaction between the anti-tumor role of omega-3 PUFAs and the DNA demethylation pathway and thus provide a firm foundation for deepening our understanding on anticancer mechanism of omega-3 PUFAs. Methods: Colorectal Cancer (CRC) model rats were induced to generate tumor by N-methyl-N-nitrosourea and their counterparts treated with omega-3 PUFAs during the induction. The blood samples from different treatment groups of rats [Normal Control group (NC), colorectal cancer model group (CRC) and omega-3 PUFAs Medication Group (MG)] were used as experimental materials. Genomic 5-hydroxymethylocytosine (5hmC) content was quantified using LC-MS/MS, and the expression of ten-eleven translocation dioxygenase 1 (TET1), catalyzing the generation of 5hmC, was also evaluated by quantitative real-time PCR. Results: We observed lower tumor incidence and small tumor size in MG group when compared with CRC group, supporting the effective anticancer role of omega-3 PUFAs. Due to the formation of CRC, 5hmC level was dramatically dropped in CRC group when compared with the NC group. Notably, 5hmC percentage in MG group remarkably increased close to NC group and was significantly higher than that in the CRC group. Consistent alteration pattern of TET1 expressions in mRNA was also observed in the tested groups of rats. Conclusion: The anticancer effect of omega-3 PUFAs was positively correlated with global 5hmC accumulation and TET1 expression, suggesting DNA hydroxymethylation pathway was factually involved in the anticancer process of omega-3 PUFAs.

Background: To explore the cytotoxic and apoptotic activity of the pierisin-6 protein in HPV HeLa and HepG2 cell lines. Methods: In this study, isolation, and purification of cytotoxic Prierisin-6 from the larvae of Pieris napi by affinity column chromatography techniques. Characterization of full-length mRNA of pierisin-6 gene was performed using 3’/5’ RACE PCR. The quantitative RT-PCR used to study the developmental stage-specific expression of pierisin-6 mRNA. The most effective concentration of Pierisin-6 protein was determined by measuring cell proliferation. Apoptosis was assessed using AO/Et-Br, Propidium Iodide, and Rhodamine 123 assays, whereas protein levels of caspase 3, cytochrome C were evaluated by ELISA method. Pierisin-6 induced cell cycle arrest was determined using Propidium iodide by FACS. Results: In this study, Pierisin-6, a novel apoptotic protein was found to have cytotoxicity against HeLa, HepG2 human cancer cell lines and L-132 human lung epithelial cell line. Among the target cells, HeLa was the most sensitive to Pierisin-6. Flow cytometry analysis confirms an increased percentage of apoptotic cells in sub G1 phase and cell cycle arrest at S phase. Alteration in the transmembrane potential of mitochondria, Cytochrome c released from the mitochondrial membrane, and caspase substrate assay demonstrated the cleavage of Ac- DEVD-pNA signifying the activation of Caspase-3. These findings suggested that Pierisin-6 significantly induce apoptosis in HeLa and HepG2 cells and is attributed mainly through a mitochondrial pathway by activation of caspases. The developmental and stage-specific expression of pierisin-6 mRNA was one thousand-fold increased from second to third instar larvae and gradually declined before pupation. Conclusion: Pierisin-6 represents a promising therapeutic approach for liver cancer patients.

Background: Cancer can be considered as a disease in which normal cells start behaving badly, multiplying uncontrollably, ignoring signals to stop and accumulating to form a mass that is generally termed as a tumor. Apoptosis or programmed cell death is a physiological process that enables organisms to control their cell numbers in many developmental and physiological settings and to eliminate unwanted cells and it plays essential role in chemotherapy-induced tumor-cell killing. The correct balance between apoptosis and inhibition of apoptosis is important in animal development as well as in tissue homeostasis. The aim of this paper is to introduce the readers about the design strategy and synthesis of effective cytotoxic and apoptotic inducing agents based on benzo[d]imidazo[2,1-b]thiazole scaffold. Methods: Benzo[d]imidazo[2,1-b]thiazole-propenone conjugates were synthesized by the condensation of 7- methoxy-2-(aryl)benzo[d]imidazo[2,1-b]thiazol-3-yl)prop-2-yn-1-ones with aryl/hetero aryl amines in ethanol at room temperature. These in turn were obtained from 7-methoxy-2-(aryl)benzo[d]imidazo[2,1-b]thiazole-3- carbaldehydes on treatment with ethynylmagnesium bromide followed by oxidation. Results: 3-Arylaminopropenone linked 2-arylbenzo[d]imidazo[2,1-b]thiazole conjugates prepared in this investigation exhibited significant cytotoxic activity and arrested HeLa cancer cells in G1 phase. The treatment of the conjugate led to 40% of loss of mitochondrial membrane potential (DΨm) in HeLa cells and 4 fold increase in the levels of reactive oxygen species (ROS). In addition, it induces apoptosis in HeLa cells, this was examined by the wound healing assay, Actin filaments and Hoechst staining assay. Conclusion: The encouraging biological profile exhibited by these 3-arylaminopropenone 2-aryl linked benzo[d]imidazo[2,1-b]thiazole conjugates demonstrate that they have the potential to be developed as a lead by further structural modifications to obtain potential chemotherapeutic agents that are likely to target the HeLa cancer cells.

Background: Berberine and cinnamic acid are natural compounds that exhibit potent anticancer activities through distinct molecular mechanisms. Objective: In the present study, we aimed to investigate the proapoptotic potential of cinnamic acid and berberine in cancer cells by examining their effect on the expression of proapoptotic and antiapoptotic genes. Moreover, the effects of berberine and cinnamic acid on the antitumor activity of cisplatin were investigated in Ehrlich solid tumor-bearing mice. Methods: For the study, 90 male mice were inoculated intramuscularly with Ehrlich ascites tumor cells (2.5 × 106/mouse), and then on day 4, mice were randomly divided into six experimental groups (group 1-untreated Ehrlich solid tumor (EST), group 2-EST treated CDDP, group 3-EST treated CA, group 4-EST treated BER, group 5-EST treated CA + CDDP, and group 6-EST treated BER + CDDP). Results: The results showed that berberine and cinnamic acid significantly decreased tumor growth and tumor volume (-74.8 and -75.5%, respectively) both as single agents and in combination with cisplatin. Moreover, both berberine and cinnamic acid increased the ratio of tumor growth inhibition (-91.5 and -92.6%, respectively), mean survival time (61.5 and 26 days, respectively), and percentage increase in lifespan (559 and 263%, respectively) of the treated mice. Our results also showed that both berberine and cinnamic acid-induced apoptosis by increasing the Bax/Bcl-2 ratio (74.1 and 45.1, respectively) and caspase-3 expression (14.3- and 11.6-fold increase, respectively). Additionally, berberine and cinnamic acid decreased oxidative stress markers, as shown by the decrease in lipid peroxidation and nitric oxide levels and an increase in reduced glutathione level. Conclusion: These results suggest that berberine and cinnamic acid have potential as antitumor and antioxidant agents derived from natural sources, which could be used alone or in combination with regular chemotherapeutic agents, such as cisplatin. These effects could be attributed to the proapoptotic activity of berberine and cinnamic acid.

Objective: The aim of this study is to investigate the inhibitory effect of camptothecin derivative 3j on Non-Small Cell Lung Cancer (NSCLCs) cells and the potential anti-tumor mechanisms. Background: Camptothecin compounds are considered as the third largest natural drugs which are widely investigated in the world and they suffered restriction because of serious toxicity, such as hemorrhagic cystitis and bone marrow suppression. Methods: Using cell proliferation assay and S180 tumor mice model, a series of 20(S)-O-substituted benzoyl 7- ethylcamptothecin compounds were screened and evaluated the antitumor activities in vitro and in vivo. Camptothecin derivative 3j was selected for further study using flow cytometry in NSCLCs cells. Cell cycle related protein cyclin A2, CDK2, cyclin D and cyclin E were detected by Western Blot. Then, computer molecular docking was used to confirm the interaction between 3j and Topo I. Also, DNA relaxation assay and alkaline comet assay were used to investigate the mechanism of 3j on DNA damage. Results: Our results demonstrated that camptothecin derivative 3j showed a greater antitumor effect in eleven 20(S)-O-substituted benzoyl 7-ethylcamptothecin compounds in vitro and in vivo. The IC50 of 3j was 1.54± 0.41 μM lower than irinotecan with an IC50 of 13.86±0.80 μM in NCI-H460 cell, which was reduced by 8 fold. In NCI-H1975 cell, the IC50 of 3j was 1.87±0.23 μM lower than irinotecan (IC50±SD, 5.35±0.38 μM), dropped by 1.8 fold. Flow cytometry analysis revealed that 3j induced significant accumulation in a dose-dependent manner. After 24h of 3j (10 μM) treatment, the percentage of NCI-H460 cell in S-phase significantly increased (to 93.54 ± 4.4%) compared with control cells (31.67 ± 3.4%). Similarly, the percentage of NCI-H1975 cell in Sphase significantly increased (to 83.99 ± 2.4%) compared with control cells (34.45 ± 3.9%) after treatment with 10μM of 3j. Moreover, increased levels of cyclin A2, CDK2, and decreased levels of cyclin D, cyclin E further confirmed that cell cycle arrest was induced by 3j. Furthermore, molecular docking studies suggested that 3j interacted with Topo I-DNA and DNA-relaxation assay simultaneously confirmed that 3j suppressed the activity of Topo I. Research on the mechanism showed that 3j exhibited anti-tumour activity via activating the DNA damage response pathway and suppressing the repair pathway in NSCLC cells. Conclusion: Novel camptothecin derivative 3j has been demonstrated as a promising antitumor agent and remains to be assessed in further studies.

Background: Novel derivatives of benzo[b]furan were found to be highly toxic towards human chronic myelogenous (K562), acute myelogenous (HL-60) and acute lymphoblastic (MOLT-4) leukemia cells. Objective: The objective was the characterization of the biological activity of novel benzofurans (influence on apoptosis, mitogen-activated protein kinases and on the cell cycle). Cellular protein(s) targeted by test benzofurans and mechanism of action were identified. Methods: The methods utilized in the study were chemical synthesis, fluorescence assays, flow cytometry, gene expression by DNA microarray and real-time RT-PCR, western blotting, cytotoxicity assays, pull-down assay, mass spectroscopy, in vitro polymerization of tubulin, molecular docking. Results: 1,1'-[3-(bromomethyl)-5,6- dimethoxy-1-benzofuran-2,7-diyldiethanone (1) and methyl 4-bromo-6- (dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylate (2) induced apoptosis in K562 and MOLT-4 cells. The profiling of gene expression revealed that 1 and 2 increased the expression of proapoptotic genes involved in both receptor (TNFRSF 10A, TNFRSF 10B, CASP8) and mitochondrial (BAX, BID, NOXA, APAF1) pathways of apoptosis. Test benzo[b]furans activated c-Jun N-terminal kinase (JNK) and p38 kinase in K562 cells. Tubulin was identified as a protein target for benzo[b]furans in pull-down experiments with biotinylated 2. Test benzo[b]furans inhibited polymerization of tubulin monomers in vitro, decreased the level of cellular microtubules and arrested cells in a G2/M phase. Molecular docking suggests that benzo[b]furans 1 and 2 bind to tubulin via colchicine binding pocket and the complex is stabilized mainly by hydrophobic interactions. Conclusion: Novel benzo[b]furans with anti-microtubule activity were identified. They induce apoptosis in cancer cells and cause G2/M cell cycle arrest. Biological activity of 1 and 2 makes them potential lead compounds for development as anticancer drugs.

Background: BRN2 transcription factor is associated with the development of malignant melanoma. The cytotoxic activities and cell death mechanism against B16F10-Nex2 cells were determined with synthetic peptide R18H derived from the POU domain of the BRN2 transcription factor. Objective: To determine the cell death mechanisms and in vivo activity of peptide R18H derived from the POU domain of the BRN2 transcription factor against B16F10-Nex2 cells. Methods: Cell viability was determined by the MTT method. C57Bl/6 mice were challenged with B16F10-Nex2 cells and treated with R18H. To identify the type of cell death, we used TUNEL assay, Annexin V and PI, Hoechst, DHE, and determination of caspase activation and cytochrome c release. Transmission electron microscopy was performed to verify morphological alterations after peptide treatment. Results: Peptide R18H displayed antitumor activity in the first hours of treatment and the EC50% was calculated for 2 and 24h, being 0.76 ± 0.045 mM and 0.559 ± 0.053 mM, respectively. After 24h apoptosis was evident, based on DNA degradation, chromatin condensation, increase of superoxide anion production, phosphatidylserine translocation, activation of caspases 3 and 8, and release of extracellular cytochrome c in B16F10-Nex2 cells. The peptide cytotoxic activity was not affected by necroptosis inhibitors and treated cells did not release LDH in the extracellular medium. Moreover, in vivo antitumor activity was observed following treatment with peptide R18H. Conclusion: Peptide R18H from BRN2 transcription factor induced apoptosis in B16F10-Nex2 and displayed antitumor activity in vivo.

Background: Our previous study successfully identified that 3,3′-Dimethylquercetin (DMQ) acted as a potent anticancer agent against human colon cancer cell lines RKO. Thus, this study was conducted to investigate the underlying mechanism by which DMQ displayed inhibitory activity in RKO cells. Methods: Flow cytometry was used to evaluate the effect of DMQ on the cell cycle arrest, as well as the mitochondrial membrane potential in RKO cells. DAPI staining and DNA fragmentation ladder assays were performed to assess the apoptosis inducing activity of DMQ. Furthermore, western blot analysis was conducted to examine the expression of related proteins responsible for the cell cycle arrest and apoptosis. Results: Treatment with DMQ caused a significant increase in the fraction of G2/M cells, and induced remarkable apoptosis. Furthermore, western blot analysis showed that DMQ arrested cells at G2/M checkpoint by down-regulation of cyclin B1, cdc2 and cdc25c and up-regulation of p21, and induced cell apoptosis via affecting the ratio of Bax/Bcl-2, causing loss of the mitochondrial membrane potential and enhancing the expression of cleaved caspase-9 (C-caspase-9) and cleaved caspase-3 (C-caspase-3). Conclusion: These data showed that DMQ could suppress RKO cell growth by arresting RKO cells at G2/M checkpoint and inducing mitochondria-dependent cell apoptosis. Our findings shed light on the potential use of DMQ as a chemotherapeutic agent for CRC.

Background/Objective: Vernonia zeylanica (L) less is an endemic plant to Sri Lanka. The present study was designed to isolate potential cytotoxic compound/s from chloroform and ethyl acetate extracts of V. zeylanica by bio-activity guided isolation and to evaluate its anti-proliferative effects in three breast cancer phenotypes (MCF -7, MDA-MB-231, SKBR-3). Methods: Combined chloroform and ethyl acetate extracts were subjected to chromatographic separations to isolate a compound (1) and the structure of the isolated compound was elucidated using 1H, 13C and mass spectroscopic techniques. Cytotoxic effects of the compound were evaluated by the sulforhodamine B (SRB) and the MTT (3- (4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. Effects of the compound on apoptosis were evaluated by fluorescent microscopy, caspase 3/7 activation, DNA fragmentation and real time PCR. Effects of the compound on the expression of heat shock protein complex were also evaluated by real time PCR and immunofluorescence. Results: Isolated compound was identified as a new sesquiterpene lactone (vernolactone). The compound mediated significant cytotoxic effects in SKBR-3 and MDA-MB-231 breast cancer cells, with little effect in MCF-7 and normal mammary epithelial MCF-10A cells. Morphological changes, DNA fragmentation, increased caspase 3/7 activities and up-regulation of p53, Bax and down regulation of Survivin confirmed the proapoptotic effects of the compound. Significant inhibition of HSP complex related genes were also observed in SKBR-3 and MDA-MB-231 breast cancer cells. Conclusion: Overall results indicate that vernolactone can mediate its cytotoxic effects via apoptosis and modulating the HSP complex.

Background: In this era of science, cancer is a black dot on the face of humankind. Consequently, the search of promising anticancer agents continues. Aims: Here we designed and synthesized new N-substituted rhodanines (RD1-7), evaluated their multispectroscopic interaction with calf thymus DNA, in silico and anticancer studies against MDA-MB-231cancer cell line. Methods: By MTT assay rhodanine RD1 was found to be the most potent with IC50 value of 72.61 μM. In addition, DNA binding studies (UV-vis and fluorescence) revealed strong binding affinity of RD1-7 with DNA (Kb in the range of 1.5-7.4 x 105 M-1). Moreover, molecular docking study, experimental DNA binding and anticancer studies are all well agreed to each other. Results: It was observed that H-bonding and hydrophobic attractions were responsible for stability of DNAcompound adducts. Besides, the reported rhodanines (RD1-7) were found as minor groove binders of DNA. Concisely, RD1-7 indicated promising pharmacological properties and hence, shows auspicious future for the development of novel anticancer agents. Conclusion: The reported rhodanines showed excellent anticancer properties. Therefore, the described rhodanines may be used as potential anticancer agents in the future.