Runx1-R188Q germ line mutation induces inflammation and predisposition to hematologic malignancies in mice.

Germ line mutations in the RUNX1 gene cause familial platelet disorder (FPD), an inherited disease associated with lifetime risk to hematopoietic malignancies (HM). Patients with FPD frequently show clonal expansion of premalignant cells preceding HM onset. Despite the extensive studies on the role of RUNX1 in hematopoiesis, its function in the premalignant bone marrow (BM) is not well-understood. Here, we characterized the hematopoietic progenitor compartments using a mouse strain carrying an FPD-associated mutation, Runx1R188Q. Immunophenotypic analysis showed an increase in the number of hematopoietic stem and progenitor cells (HSPCs) in the Runx1R188Q/+ mice. However, the comparison of Sca-1 and CD86 markers suggested that Sca-1 expression may result from systemic inflammation. Cytokine profiling confirmed the dysregulation of interferon-response cytokines in the BM. Furthermore, the expression of CD48, another inflammation-response protein, was also increased in Runx1R188Q/+ HSPCs. The DNA-damage response activity of Runx1R188Q/+ hematopoietic progenitor cells was defective in vitro, suggesting that Runx1R188Q may promote genomic instability. The differentiation of long-term repopulating HSCs was reduced in Runx1R188Q/+ recipient mice. Furthermore, we found that Runx1R188Q/+ HSPCs outcompete their wild-type counterparts in bidirectional repopulation assays, and that the genetic makeup of recipient mice did not significantly affect the clonal dynamics under this setting. Finally, we demonstrate that Runx1R188Q predisposes to HM in cooperation with somatic mutations found in FPDHM, using 3 mouse models. These studies establish a novel murine FPDHM model and demonstrate that germ line Runx1 mutations induce a premalignant phenotype marked by BM inflammation, selective expansion capacity, defective DNA-damage response, and predisposition to HM.

CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFß-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

Regioselective synthesis and biological studies of novel 1-aryl-3, 5-bis (het) aryl pyrazole derivatives as potential antiproliferative agents.

Pyrazole moiety represents an important category of heterocyclic compound in pharmaceutical and medicinal chemistry. The novel 1-aryl-3, 5-bis (het) aryl pyrazole derivatives were synthesized with complementary regioselectivity. The chemical structures were confirmed by IR, 1H NMR, 13C NMR, and mass spectral analysis. The chemical entities were screened in various cancer cell lines to assess their cell viability activity. Results showed that the compound 3-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl) pyridine (5d) possessed maximum cytotoxic effect against breast cancer and leukemic cells. The cytotoxicity was confirmed by live-dead cell assay and cell cycle analysis. Mitochondrial membrane potential, Annexin V-FITC staining, DNA fragmentation, Hoechst staining, and western blot assays revealed the ability of compound 5d to induce cell death by activating apoptosis in cancer cells. Thus, the present study demonstrates that compound 5d could be an attractive chemical entity for the development of small molecule inhibitors for treatment of leukemia and breast cancer.

A novel inhibitor of BCL2, Disarib abrogates tumor growth while sparing platelets, by activating intrinsic pathway of apoptosis.

Antiapoptotic protein BCL2, serves as an excellent target for anticancer therapy owing to its increased level in cancers. Previously, we have described characterization of a novel BCL2 inhibitor, Disarib, which showed selective cytotoxicity in BCL2 'high' cancer cells and CLL patient cells. Here, we have investigated the mechanism of Disarib-induced cytotoxicity, and compared its efficacy with a well-established BCL2 inhibitor, ABT199. We show that Disarib administration caused tumor regression in mouse allograft and xenograft models, exhibited platelet sparing property and did not exhibit significant side effects. Importantly, comparison between Disarib and ABT199, revealed higher efficacy for Disarib in mouse tumor model and cancer cell lines. Disarib induced cell death by activating intrinsic apoptotic pathway. Interestingly, Disarib showed synergism with paclitaxel, suggesting its potential for combination therapy. Thus, we provide mechanistic insights into the cell death pathways induced by Disarib, report that Disarib exhibited better effect than currently used ABT199 and demonstrate its combinatorial potential with paclitaxel.

A Novel Resveratrol Based Tubulin Inhibitor Induces Mitotic Arrest and Activates Apoptosis in Cancer Cells.

Resveratrol is one of the most widely studied bioactive plant polyphenols which possesses anticancer properties. Previously we have reported synthesis, characterization and identification of a novel resveratrol analog, SS28. In the present study, we show that SS28 induced cytotoxicity in several cancer cell lines ex vivo with an IC50 value of 3-5 µM. Mechanistic evaluation of effect of SS28 in non-small cell lung cancer cell line (A549) and T-cell leukemic cell line (CEM) showed that it inhibited Tubulin polymerization during cell division to cause cell cycle arrest at G2/M phase of the cell cycle at 12-18 h time period. Immunofluorescence studies confirmed the mitotic arrest upon treatment with SS28. Besides, we show that SS28 binds to Tubulin with a dissociation constant of 0.414 ± 0.11 µM. Further, SS28 treatment resulted in loss of mitochondrial membrane potential, activation of Caspase 9 and Caspase 3, leading to PARP-1 cleavage and finally cell death via intrinsic pathway of apoptosis. Importantly, treatment with SS28 resulted in regression of tumor in mice. Hence, our study reveals the antiproliferative activity of SS28 by disrupting microtubule dynamics by binding to its cellular target Tubulin and its potential to be developed as an anticancer molecule.

Identification of a novel BCL2-specific inhibitor that binds predominantly to the BH1 domain.

The antiapoptotic protein BCL2 is overexpressed in several cancers and contributes to prolonged cell survival and chemoresistance, lending itself as an excellent target for cancer therapy. Here, we report the design, synthesis, and characterization of Disarib, a novel BCL2 inhibitor. Disarib showed selective cytotoxicity in BCL2 high cancer cell lines, and CLL patient primary cells, as compared to BCL2 low cell lines. BCL2 knockdown in cells rendered remarkable resistance to Disarib, while sensitivity was regained upon its ectopic expression, establishing target specificity. In silico, biochemical and biophysical studies demonstrated strong affinity of Disarib to BCL2, but not to other antiapoptotic BCL2 family members viz., BCL-xL, BCL2A1 etc. Interestingly, biophysical studies showed that BH1 domain deletion mutant demonstrated ~ 67-fold reduction in BCL2-Disarib interaction, while it was only ~ 20-fold in the case of BH3 deletion mutant, suggesting predominant involvement of the BH1 domain for Disarib binding. Thus, we report identification of a novel BCL2 inhibitor with a unique mechanism of BCL2 inhibition, as opposed to the well-studied BH3 domain targeting.

Induction of apoptosis and downregulation of ERα in DMBA-induced mammary gland tumors in Sprague-Dawley rats by synthetic 3,5-disubstituted isoxazole derivatives.

Isoxazole derivatives are an important group of chemotherapeutic prototypes. In the current study, we have synthesized few isoxazole derivatives and tested them for their antiproliferative properties in cancer cell lines such as MCF7 and HeLa. The lead compound, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)isoxazole (2b), showed considerable inhibition of proliferation of MCF7 and HeLa cells with the IC50 values of 19.5 and 39.2 µM, respectively. Cell cycle analyses and annexin-FITC staining in 2b-treated breast adenocarcinoma cells (MCF7) showed increased sub-G1 population and apoptosis. Furthermore, we tested the tumor inhibitory effect of 2b and estrogen receptor expression profile in DMBA-induced mammary tumors in Sprague-Dawley rats. The gross morphology of tumor studies was investigated by histopathology and ERα protein expression was evaluated by immunohistochemistry, which showed tumor regression and downregulation of ERα in tumor cells. The present results implicate that compound 2b could be used for the further derivatization for the treatment of breast cancer.

Combinatorial Study of a Novel Poly (ADP-ribose) Polymerase Inhibitor and an HDAC Inhibitor, SAHA, in Leukemic Cell Lines.

BACKGROUND: Cancer is a multifactorial disease, which makes it difficult to cure. Since more than one defective cellular component is often involved during oncogenesis, combination therapy is gaining prominence in the field of cancer therapeutics. OBJECTIVE: The purpose of this study was to investigate the combinatorial effects of a novel PARP inhibitor, P10, and HDAC inhibitor, SAHA, in leukemic cells. METHODS: Combinatorial effects of P10 and SAHA were tested using propidium iodide staining in different leukemic cells. Further, flowcytometry-based assays such as calcein-AM/ethidium homodimer staining, annexin-FITC/PI staining, and JC-1 staining were carried out to elucidate the mechanism of cell death. In addition, cell-cycle analysis, immunocytochemistry studies, and western blotting analysis were conducted to check the combinatorial effect in Nalm6 cells. RESULTS: Propidium iodide staining showed that P10 in combination with SAHA induced cell death in Nalm6 cells, in which PARP expression and activity is high with a combination index of <0.2. Annexin-FITC/PI staining, JC-1 staining, and other biochemical assays revealed that P10 in combination with SAHA induced apoptosis by causing a change in mitochondrial membrane potential in >65 % cells. Importantly, combinatorial treatment induced S phase arrest in 40-45 % cells due to DNA damage and plausible replicative stress. Finally, we demonstrated that treatment with P10 led to DNA strand breaks, which were further potentiated by SAHA (p < 0.01), leading to activation of apoptosis and increased cell death in PARP-positive leukemic cells. CONCLUSIONS: Our study reveals that coadministration of PARP inhibitor with SAHA could be used as a combination therapy against leukemic cells that possess high levels of intrinsic PARP activity.

Quercetin, a Natural Flavonoid Interacts with DNA, Arrests Cell Cycle and Causes Tumor Regression by Activating Mitochondrial Pathway of Apoptosis.

Naturally occurring compounds are considered as attractive candidates for cancer treatment and prevention. Quercetin and ellagic acid are naturally occurring flavonoids abundantly seen in several fruits and vegetables. In the present study, we evaluate and compare antitumor efficacies of quercetin and ellagic acid in animal models and cancer cell lines in a comprehensive manner. We found that quercetin induced cytotoxicity in leukemic cells in a dose-dependent manner, while ellagic acid showed only limited toxicity. Besides leukemic cells, quercetin also induced cytotoxicity in breast cancer cells, however, its effect on normal cells was limited or none. Further, quercetin caused S phase arrest during cell cycle progression in tested cancer cells. Quercetin induced tumor regression in mice at a concentration 3-fold lower than ellagic acid. Importantly, administration of quercetin lead to ~5 fold increase in the life span in tumor bearing mice compared to that of untreated controls. Further, we found that quercetin interacts with DNA directly, and could be one of the mechanisms for inducing apoptosis in both, cancer cell lines and tumor tissues by activating the intrinsic pathway. Thus, our data suggests that quercetin can be further explored for its potential to be used in cancer therapeutics and combination therapy.

Platelet protective efficacy of 3,4,5 trisubstituted isoxazole analogue by inhibiting ROS-mediated apoptosis and platelet aggregation.

Thrombocytopenia is a major hematological concern in oxidative stress-associated pathologies and chronic clinical disorders, where premature platelet destruction severely affects the normal functioning of thrombosis and hemostasis. In addition, frequent exposure of platelets to chemical entities and therapeutic drugs immensely contributes in the development of thrombocytopenia leading to huge platelet loss, which might be fatal sometimes. Till date, there are only few platelet protective molecules known to combat thrombocytopenia. Hence, small molecule therapeutics are extremely in need to relieve the burden on limited treatment strategies of thrombocytopenia. In this study, we have synthesized a series of novel 3,4,5 trisubstituted isoxazole derivatives, among which compound 4a [4-methoxy-N'-(5-methyl-3-phenylisoxazole-4-carbonyl) benzenesulfonohydrazide] was found to significantly ameliorate the oxidative stress-induced platelet apoptosis by restoring various apoptotic markers such as ROS content, cytosolic Ca(2+) levels, eIF2-α phosphorylation, mitochondrial membrane depolarization, cytochrome c release, caspase activation, PS externalization, and cytotoxicity markers. Additionally, compound 4a dose dependently inhibits collagen-induced platelet aggregation. Hence, compound 4a can be considered as a prospective molecule in the treatment regime of platelet activation and apoptosis and other clinical conditions of thrombocytopenia. Further studies might ensure the use of compound 4a as a supplementary therapeutic agent to treat, thrombosis and CVD-associated complications. Over all, the study reveals a platelet protective efficacy of novel isoxazole derivative 4a with a potential to combat oxidative stress-induced platelet apoptosis.

Induction Chemotherapy with Cisplatin and 5-Fluorouracil in Advanced Head and Neck Cancers: A Short Term Response Evaluation.

BACKGROUND: Considering the uprising number of Head and neck cancer in the state with limited options of medical and surgical treatment, the focus of this study involved on chemotherapy in advanced Head and neck cancers. The aim of this study was to evaluate the efficacy and toxicity of combination of Cisplatin and 5-Fluorouracil (PF) as induction chemotherapy in patients in locally advanced squamous cell cancer of head and neck. MATERIALS AND METHODS: Forty four patients with previously untreated stage III -IV advanced and inoperable cases were included in this prospective study. Induction chemotherapy consisted of 3 cycles of Cisplatin 100mg/mt(2) as infusion on day 1, 5-Fluorouracil of 750mg/mt(2) on day 2, 5-Fluorouracil of 1000mg/mt2 as infusion on day 3 in an inpatient basis. Cycles were repeated with an interval of 21 days. Patients were evaluated within a period of 3 weeks at the end of completion of third cycle of chemotherapy. Post chemotherapy local therapy was individualized based on the response, site and stage of the tumour. RESULTS: Out of 44 eligible and evaluable patients, major dominance was noted in male group constituting 68%. After induction chemotherapy 58.8% of stage III experienced stable response, & 44% had partial response. In stage IV, 44% showed a stable response and 33.3% had partial response. But in comparison to primary tumour response and nodal response, which had a significant clinical response, the overall response of malignancy with respect to stage and site specificity was clinically insignificant. Moderate adverse reaction was noted in 47.6% and 42.1% had mild reactions. Majority of patients experienced grade 3 adverse events, of which anaemia in females and leucopenia in males pre-dominated. CONCLUSION: With the use of cisplatin and 5-FU as induction chemotherapy agents in advanced and inoperable squamous cell carcinoma of head and neck, a distinct benefit was seen in stabilizing the tumour from progression. But achieving a significant complete response to the same is of faint possibility. An alternate multidrug regimen or multimodality treatment would be ideal to gain the optimum results from induction agents. Toxicity related to chemotherapy usually is transient at therapeutic doses, and can be controlled by adequate prophylactic measures.

Antiproliferative and tumor inhibitory studies of 2,3 disubstituted 4-thiazolidinone derivatives.

4-Thiazolidinone derivatives were synthesized using T3P®-DMSO media as a cyclodehydrating agent. All the molecules were tested for their cytotoxicity against leukemic cell lines. The compound 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)thiazolidin-4-one (4e) with electron donating substituent at para position of phenyl ring displayed considerable cytotoxicity against Reh and Nalm6 cells with an IC50 value of 11.9 and 13.5 µM, respectively. Furthermore, the compound 4e tested for tumor regression studies induced by EAC in Swiss albino mouse. Both in vitro and in vivo results suggested significant antiproliferative activity of compound 4e in Reh cells and mouse tumor tissue treated with compound 4e showed multifocal areas of necrosis and numerous number of apoptotic cells.

Synthesis and antiproliferative activity of imidazo[2,1-b][1,3,4]thiadiazole derivatives.

A series of 2,5,6-substituted imidazo[2,1-b][1,3,4]thiadiazole derivatives have been prepared and were tested for antiproliferative activity on cancer cells at the National Cancer Institute. Results showed that molecules with a benzyl group at position 2, exhibited an increase in activity for the introduction of a formyl group at the 5 position. The compound 2-benzyl-5-formyl-6-(4-bromophenyl)imidazo[2,1-b][1,3,4]thiadiazole 22 has been chosen for understanding the mechanism of action by various molecular and cellular biology studies. Results obtained from cell cycle evaluation analysis, analysis of mitochondrial membrane potential and Annexin V-FITC by flow cytometric analysis, ROS production and expression of apoptotic and DNA-repair proteins suggested that compound 22 induced cytotoxicity by activating extrinsic pathway of apoptosis, however, without affecting cell cycle progression.

Sapodilla plum (Achras sapota) induces apoptosis in cancer cell lines and inhibits tumor progression in mice.

Intake of fruits rich in antioxidants in daily diet is suggested to be cancer preventive. Sapota is a tropical fruit grown and consumed extensively in several countries including India and Mexico. Here we show that methanolic extracts of Sapota fruit (MESF) induces cytotoxicity in a dose-dependent manner in cancer cell lines. Cell cycle analysis suggested activation of apoptosis, without arresting cell cycle progression. Annexin V-propidium iodide double-staining demonstrated that Sapota fruit extracts potentiate apoptosis rather than necrosis in cancer cells. Loss of mitochondrial membrane potential, upregulation of proapoptotic proteins, activation of MCL-1, PARP-1, and Caspase 9 suggest that MESF treatment leads to activation of mitochondrial pathway of apoptosis. More importantly, we show that MESF treatment leads to significant inhibition of tumor growth and a 3-fold increase in the life span of tumor bearing animals compared to untreated tumor mice.

2-(4-Chlorobenzyl)-6-arylimidazo[2,1-b][1,3,4]thiadiazoles: synthesis, cytotoxic activity and mechanism of action.

The cytotoxic activity of a new series of 2-(4'-chlorobenzyl)-5,6-disubstituted imidazo[2,1-b][1,3,4]thiadiazoles against different human and murine cancer cell lines is reported. Among the tested compounds, two derivatives namely 2-(4-chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehyde 4i and 2-(4-chlorobenzyl)-6-(2-oxo-2H-chromen-3-yl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl thiocyanate 5i emerged as the most potent against all the cell lines. To investigate the mechanism of action, we selected compounds 4i for cell cycle study, analysis of mitochondrial membrane potential and Annexin V-FITC flow cytometric analysis and DNA fragmentation assay. Results showed that 4i induced cytotoxicity by inducing apoptosis without arresting the cell cycle.

Synthesis and antiproliferative effect of novel 4-thiazolidinone-, pyridine- and piperazine-based conjugates on human leukemic cells.

The present work reveals the synthesis and antiproliferative effect of a series of 2, 3 disubstituted 4-thiazolidinone analogues on human leukemic cells. The chemical structures of newly synthesized compounds were confirmed by IR, (1)H NMR, (13)C NMR and mass spectral analysis. Compound methyl 3-methoxy-4-(4-oxo-3-(5-(piperazin-1-yl)pyridin-2-yl)thiazolidin-2-yl)benzoate (5) displayed potent activity (IC509.71, 15.24 and 19.29 µM) against Nalm6, K562, Jurkat cells. Cell cycle analysis and mitochondrial membrane potential further confirmed that compound 5 is cytotoxic and able to induce cell death.

Oral Microflora: A Comparative Study in HIV and Normal Patients.

The study was designed to compare the oral microbiota in normal and HIV-infected individuals. The study tries to establish a significant shift in oral microflora in HIV-infected patients. Antibiotic sensitivity testing was performed to establish any rise in resistance against the antibiotics. It was a two and half year prospective study conducted in a tertiary care centre. The study group consisted of eighty subjects divided into two groups of control and HIV. The age range for this group was 9-75 years. The mean age in this group was 39.7 years. The male:female ratio was 2.75:1. Tuberculosis was the most common opportunistic infection in patients with HIV infection. The most common commensal micro organism isolated was the Viridans streptococci in 60% followed by Streptococcus pneumoniae in 23.33%. HIV Group: The most common commensal micro organism isolated was the Viridans streptococci in 42%; this was followed by the Micrococci spp. in 22% cases. S. pneumoniae was isolated in 6% of cases. The colony count for Viridans streptococci showed a heavy growth in 55.56% of cases in controls whereas the same in HIV group was 62.5%. Micrococcus spp. was isolated from 11 subjects in HIV group while it was not isolated from the controls. 50% subjects in the HIV group showed a heavy growth of Klebsiella spp. whereas controls showed only moderate and scanty growth. In patients with CD4+ T cell count less than 50 cells/µl we found a heavy colonization of the oral cavity with Micrococcus spp., Acinetobacter and Klebsiella spp. Viridans streptococcus was not isolated in any of the patients with CD4+ T cell count less than 50 cells/µl. As CD4+ T cells counts improved to 51-100 cells/µl Viridans streptococcus colonies returned and 37.5% patients showed a heavy growth. Micrococcus spp. colonies were isolated till the CD4+ T cells improved up to 300 cells/µl. At counts > 300 cells/µl the oral microbiota became comparable to that of the controls. Many of the opportunistic infections in HIV are caused by commensal bacteria which are otherwise harmless in a normal individual. Our study is unique in that such a study of the oral commensals in HIV patients has never been reported. We found an increased colonization of the oral cavity by Micrococcus spp. which is a normal commensal of the skin.

Cervical thymic cyst: a rare differential diagnosis in lateral neck swelling.

Introduction. Thymic cysts are among the rarest cysts found in the neck. Nests of thymic tissue may be found anywhere along the descent of the thymic primordia from the angle of the mandible to the mediastinum. Mediastinal extension is seen in 50% of cervical thymic cysts. Case Report. We report an uncommon case of a 15-year-old male, who noted a painless, growing mass on left side of his neck of one-year duration. Computerised tomographic scan showed a multiloculated fluid density lesion with enhancing septae in the left parapharyngeal space, extending from the level of mandible up to C7 vertebral level. Here, we discuss the surgical aspect, histopathology, and management of this rare lateral neck swelling. Discussion. Clinically, in most cases, cervical thymic lesions present as a unilateral asymptomatic neck mass, commonly on the left side of the neck, and 75% of patients present before 20 years of age. Conclusion. Thymic cyst should be included as differential diagnosis of cystic neck masses. Greater awareness among the pathologists may decrease misdiagnosis.