Novel amino substituted tetracyclic imidazo[4,5-b]pyridine derivatives: Design, synthesis, antiproliferative activity and DNA/RNA binding study.

A novel series of tetracyclic imidazo[4,5-b]pyridine derivatives was designed and synthesized as potential antiproliferative agents. Their antiproliferative activity against human cancer cells was influenced by the introduction of chosen amino side chains on the different positions on the tetracyclic skeleton and particularly, by the position of N atom in the pyridine nuclei. Thus, the majority of compounds showed improved activity in comparison to standard drug etoposide. Several compounds showed pronounced cytostatic effect in the submicromolar range, especially on HCT116 and MCF-7 cancer cells. The obtained results have confirmed the significant impact of the position of N nitrogen in the pyridine ring on the enhancement of antiproliferative activity, especially for derivatives bearing amino side chains on position 2. Thus, regioisomers 6, 7 and 9 showed noticeable enhancement of activity in comparison to their counterparts 10, 11 and 13 with IC50 values in a nanomolar range of concentration (0.3-0.9 µM). Interactions with DNA (including G-quadruplex structure) and RNA were influenced by the position of amino side chains on the tetracyclic core of imidazo[4,5-b]pyridine derivatives and the ligand charge. Moderate to high binding affinities (logKs = 5-7) obtained for selected imidazo[4,5-b]pyridine derivatives suggest that DNA/RNA are potential cell targets.

Targeting miRNAs by natural products: A new way for cancer therapy.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression through mRNA degradation or translation inhibition. MiRNAs play important roles in a variety of biological processes, and dysregulation of miRNA expression is highly associated with cancer development. Individual miRNA regulates multiple gene expressions, enabling them to regulate multiple cellular signaling pathways simultaneously. Hence, miRNAs could be served as cancer biomarkers for diagnosis and prognosis, and also therapeutic targets. Recently, more and more evidences showed that natural products such as paclitaxel, curcumin, resveratrol, genistein or epigallocatechin-3-gallate exert their anti-proliferative and/or pro-apoptotic effects through regulating one or more miRNAs, leading to the inhibition of cancer cell growth, induction of apoptosis or enhancement of conventional cancer therapeutic efficacy. Herein, we outlined the recent advances in the regulation of miRNAs expression by the natural products and highlight the importance of these natural drugs as a potential strategy in cancer treatment. This review will help us better understand how natural products modulate miRNAs and contribute to the development of effective and safe natural drugs for therapeutic purposes.

Targeting the estrogen receptor alpha (ERα)-mediated circ-SMG1.72/miR-141-3p/Gelsolin signaling to better suppress the HCC cell invasion.

Early studies indicated that estrogen receptor α (ERα) might impact the progression of hepatocellular carcinoma (HCC). However, the detailed mechanisms, especially its linkage to the gelsolin (GSN)-mediated cell invasion, remain unclear. Here we found that ERα could decrease HCC cell invasion via suppressing the circular RNA-SMG1.72 (circRNA-SMG1.72) expression via transcriptional regulation through directly binding to the 5' promoter region of its host gene SMG1, We showed that ERα-suppressed circ-SMG1.72 could sponge and inhibit the expression of the microRNA (miRNA, miR), miR-141-3p, which could then result in increasing the GSN messenger RNA translation via reduced miR binding to its 3' untranslated region (3'UTR). The preclinical study using an in vivo mouse model with orthotopic xenografts of HCC cells confirmed the in vitro data, and the human HCC clinical sample survey and tissue staining also confirmed the linkage of ERα/miR-141-3p/GSN signaling to the HCC progression. Together, our findings suggest that ERα can suppress HCC cell invasion via altering the ERα/circRNA-SMG1.72/miR-141-3p/GSN signaling, and targeting this newly identified signaling with small molecules may help in the development of novel therapies to better suppress the HCC progression.

Combined Scaffold Evaluation and Systems-Level Transcriptome-Based Analysis for Accelerated Lead Optimization Reveals Ribosomal Targeting Spirooxindole Cyclopropanes.

With evolutionary drug resistance impacting efforts to treat disease, the need for small molecules that exhibit novel molecular mechanisms of action is paramount. In this study, we combined scaffold-directed synthesis with a hybrid experimental and transcriptome analysis to identify bis-spirooxindole cyclopropanes that inhibit cancer cell proliferation through disruption of ribosomal function. These findings demonstrate the value of an integrated, biologically inspired synthesis and assay strategy for the accelerated identification of first-in-class cancer therapeutic candidates.

Dose enhancement effects of gold nanoparticles specifically targeting RNA in breast cancer cells.

Localization microscopy has shown to be capable of systematic investigations on the arrangement and counting of cellular uptake of gold nanoparticles (GNP) with nanometer resolution. In this article, we show that the application of specially modified RNA targeting gold nanoparticles ("SmartFlares") can result in ring like shaped GNP arrangements around the cell nucleus. Transmission electron microscopy revealed GNP accumulation in vicinity to the intracellular membrane structures including them of the endoplasmatic reticulum. A quantification of the radio therapeutic dose enhancement as a proof of principle was conducted with γH2AX foci analysis: The application of both-SmartFlares and unmodified GNPs-lead to a significant dose enhancement with a factor of up to 1.2 times the dose deposition compared to non-treated breast cancer cells. This enhancement effect was even more pronounced for SmartFlares. Furthermore, it was shown that a magnetic field of 1 Tesla simultaneously applied during irradiation has no detectable influence on neither the structure nor the dose enhancement dealt by gold nanoparticles.

Curcumin suppresses proliferation and in vitro invasion of human prostate cancer stem cells by ceRNA effect of miR-145 and lncRNA-ROR.

Many studies have demonstrated that curcumin can effectively inhibit the proliferation, invasion, and tumorigenesis of prostate cancer cells in vitro and in vivo. In this study, CD44+/CD133+ human prostate cancer stem cells (HuPCaSCs) were isolated from the prostate cancer cell lines Du145 and 22RV1. Curcumin treatment of these cells resulted in the inhibition of in vitro proliferation and invasion, and cell cycle arrest. The expression levels of cell cycle proteins (Ccnd1 and Cdk4) and stem cell markers (Oct4, CD44, and CD133) were decreased in curcumin-treated HuPCaSCs. Microarray analysis and northern blotting assays indicated that miR-145 was overexpressed in curcumin-treated HuPCaSCs. Insights of the mechanism of competitive endogenous RNAs (ceRNAs) were gained from bioinformatic analysis, bioinformatics analysis and luciferase activity assays showed that the lncRNA-ROR and Oct4 mRNA both contain miR-145 binding sites, and Oct4 and lncRNA-ROR directly compete for microRNA binding. Curcumin induced high miR-145 expression and inhibited the expression of lncRNA-ROR. The tumorigenicity of curcumin- treated HuPCaSCs in nude mice was significantly reduced. In summary, reducing the expression of endogenous lncRNA-ROR could effectively increase the available concentration of miR-145 in HuPCaSCs, where miR-145 prevents cell proliferation by decreasing Oct4 expression. In particular, we hypothesized that lncRNA-ROR may act as a ceRNA, effectively becoming a sink for miR-145, thereby activating the derepression of core transcription factors Oct4. Thus, curcumin suppresses the proliferation, in vitro invasion, and tumorigenicity of HuPCaSCs through ceRNA effect of miR-145 and lncRNA-ROR caused.

The long noncoding RNA PVT1 functions as a competing endogenous RNA by sponging miR-186 in gastric cancer.

BACKGROUND: Recent evidence has highlighted the key regulatory roles of long non-coding RNAs (lncRNAs) in tumor development and progression including gastric cancer (GC).The long non-coding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) has been identified as an oncogene in some tumors. However, the potential biological roles and regulatory mechanisms of PVT1 involved in GC remained poorly understood. METHODS: Quantitative real-time PCR (QRT-PCR) was used to determine the expression of PVT1 and miR-186 in GC tissues. The MTT cell proliferation and transwell invasion assays were used to detect the cell proliferation and invasion abilities. Western-blotting analysis was used to detect the protein expression of PCNA and HIF-1α. To understand the tumorigenic mechanism of PVT1, luciferase reporter assays were performed to evaluate whether the miR-186 was a target of PVT1 in GC cells. RESULTS: In the current study, we showed that PVT1 expression was markedly upregulated in GC tissues and cell lines, and high expression levels of PVT1 were obviously correlated with advanced tumor stage and lymph node metastasis. Further functional experiments indicated up-regulation of PVT1 promoted the GC cell proliferation and invasion, while down-regulation of PVT1 inhibited cell proliferation and invasion. In addition, PVT1 could directly interact with miR-186 in GC cells and this interaction lead to the inhibition of downstream of HIF-1α expression. CONCLUSIONS: These results suggested that PVT1 acted as a key role in GC pathogenesis and may serve as a potential therapeutic target for GC.

Synthesis, in vitro evaluation, and molecular modeling investigation of benzenesulfonimide peroxisome proliferator-activated receptors α antagonists.

Recent evidences suggest a moderate activation of Peroxisome Proliferator-Activated Receptors (PPARs) could be favorable in metabolic diseases, reducing side effects given from full agonists. PPAR partial agonists and antagonists represent, to date, interesting tools to better elucidate biological processes modulated by these receptors. In this work are reported new benzenesulfonimide compounds able to block PPARα, synthesized and tested by transactivation assays and gene expression analysis. Some of these compounds showed a dose-dependent antagonistic behavior on PPARα, submicromolar potency, different profiles of selectivity versus PPARγ, and a repressive effect on CPT1A expression. Dockings and molecular dynamics on properly selected benzenesulfonimide derivatives furnished fresh insights into the molecular determinant most likely responsible for PPARα antagonism.

Synthesis of Triphenylethylene Bisphenols as Aromatase Inhibitors That Also Modulate Estrogen Receptors.

A series of triphenylethylene bisphenol analogues of the selective estrogen receptor modulator (SERM) tamoxifen were synthesized and evaluated for their abilities to inhibit aromatase, bind to estrogen receptor α (ER-α) and estrogen receptor ß (ER-ß), and antagonize the activity of ß-estradiol in MCF-7 human breast cancer cells. The long-range goal has been to create dual aromatase inhibitor (AI)/selective estrogen receptor modulators (SERMs). The hypothesis is that in normal tissue the estrogenic SERM activity of a dual AI/SERM could attenuate the undesired effects stemming from global estrogen depletion caused by the AI activity of a dual AI/SERM, while in breast cancer tissue the antiestrogenic SERM activity of a dual AI/SERM could act synergistically with AI activity to enhance the antiproliferative effect. The potent aromatase inhibitory activities and high ER-α and ER-ß binding affinities of several of the resulting analogues, together with the facts that they antagonize ß-estradiol in a functional assay in MCF-7 human breast cancer cells and they have no E/Z isomers, support their further development in order to obtain dual AI/SERM agents for breast cancer treatment.

Comparison of protoporphyrin IX produced cell proliferation inhibition between human breast cancer MCF-7 and MDA-MB-231 cells.

Protoporphyrin IX (PpIX) is an effective hematoporphyrin derivative, widely adopted in photodynamic therapy (PDT) and sonodynamic therapy (SDT). As a sensitizer, PpIX could significantly enhance laser light or ultrasound causing tumor cell damage in PDT/SDT studies. However, the biological function of PpIX itself has not been carefully defined. Recently, studies indicate that PpIX alone can inhibit Hela cell proliferation, but the potential mechanism was unclear. Therefore, in the present study it was investigated whether the proliferation inhibition effect generally occurred in human breast cancer MCF-7 and MDA-MB-231 cells. Different sensitivities and the involved mechanisms were carefully explored. Our results show that PpIX preferentially accumulated and selectively caused cell damage in human breast cancer MCF-7 and MDA-MB-231 cells compared with mouse embryonic fibroblast NIH-3T3. In vitro, PpIX induced cell viability decrease, intracellular ROS (reactive oxygen species) generation, and DNA damage in a concentration-dependent and cell-specific manner. MCF-7 was more sensitive to PpIX than MDA-MB-231 cells at the same PpIX dose. Western blots showed obvious enhancement of P53, and PUMA in a concentration dependent manner in MCF-7 cells, but not in MDA-MB-231 cells. In cell-free system, we also found that PpIX could interact with some large biological molecules, such as calf thymus DNA, and induce hyperchromic effects in spectroscopic analysis. Our findings imply that DNA might be one of the main targets of PpIX, and PpIX alone can cause significant tumor cell damage through ROS generation, while P53 status may play an important role in these processes.

Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells

Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68±13% (P=0.002) and increased the number of apoptotic cells by 9.8±1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

Downregulation of survivin by siRNA inhibits invasion and promotes apoptosis in neuroblastoma SH-SY5Y cells.

Neuroblastoma is a solid tumor that occurs mainly in children. Malignant neuroblastomas have a poor prognosis because conventional chemotherapeutic agents are not very effective. Survivin, a member of the inhibitor of the apoptosis protein family, plays a significant role in cell division, inhibition of apoptosis, and promotion of cell proliferation and invasion. Previous studies found that survivin is highly expressed in some malignant neuroblastomas and is correlated with poor prognosis. The aim of this study was to investigate whether survivin could serve as a potential therapeutic target of human neuroblastoma. We employed RNA interference to reduce survivin expression in the human neuroblastoma SH-SY5Y cell line and analyzed the effect of RNA interference on cell proliferation and invasion in vitro and in vivo. RNA interference of survivin led to a significant decrease in invasiveness and proliferation and increased apoptosis in SH-SY5Y cells in vitro. RNA interference of survivin inhibited tumor growth in vivo by 68 ± 13% (P=0.002) and increased the number of apoptotic cells by 9.8 ± 1.2% (P=0.001) compared with negative small interfering RNA (siRNA) treatment controls. Moreover, RNA interference of survivin inhibited the formation of lung metastases by 92% (P=0.002) and reduced microvascular density by 60% (P=0.0003). Survivin siRNA resulted in significant downregulation of survivin mRNA and protein expression both in vitro and in vivo compared with negative siRNA treatment controls. RNA interference of survivin was found to be a potent inhibitor of SH-SY5Y tumor growth and metastasis formation. These results support further clinical development of RNA interference of survivin as a treatment of neuroblastoma and other cancer types.

Conjugation of a Ru(II) arene complex to neomycin or to guanidinoneomycin leads to compounds with differential cytotoxicities and accumulation between cancer and normal cells.

A straightforward methodology for the synthesis of conjugates between a cytotoxic organometallic ruthenium(II) complex and amino- and guanidinoglycosides, as potential RNA-targeted anticancer compounds, is described. Under microwave irradiation, the imidazole ligand incorporated on the aminoglycoside moiety (neamine or neomycin) was found to replace one triphenylphosphine ligand from the ruthenium precursor [(η(6)-p-cym)RuCl(PPh3)2](+), allowing the assembly of the target conjugates. The guanidinylated analogue was easily prepared from the neomycin-ruthenium conjugate by reaction with N,N'-di-Boc-N″-triflylguanidine, a powerful guanidinylating reagent that was compatible with the integrity of the metal complex. All conjugates were purified by semipreparative high-performance liquid chromatography (HPLC) and characterized by electrospray ionization (ESI) and matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR spectroscopy. The cytotoxicity of the compounds was tested in MCF-7 (breast) and DU-145 (prostate) human cancer cells, as well as in the normal HEK293 (Human Embryonic Kidney) cell line, revealing a dependence on the nature of the glycoside moiety and the type of cell (cancer or healthy). Indeed, the neomycin-ruthenium conjugate (2) displayed moderate antiproliferative activity in both cancer cell lines (IC50 ≈ 80 µM), whereas the neamine conjugate (4) was inactive (IC50 ≈ 200 µM). However, the guanidinylated analogue of the neomycin-ruthenium conjugate (3) required much lower concentrations than the parent conjugate for equal effect (IC50 = 7.17 µM in DU-145 and IC50 = 11.33 µM in MCF-7). Although the same ranking in antiproliferative activity was found in the nontumorigenic cell line (3 ≫ 2 > 4), IC50 values indicate that aminoglycoside-containing conjugates are about 2-fold more cytotoxic in normal cells (e.g., IC50 = 49.4 µM for 2) than in cancer cells, whereas an opposite tendency was found with the guanidinylated conjugate, since its cytotoxicity in the normal cell line (IC50 = 12.75 µM for 3) was similar or even lower than that found in MCF-7 and DU-145 cancer cell lines, respectively. Cell uptake studies performed by ICP-MS with conjugates 2 and 3 revealed that guanidinylation of the neomycin moiety had a positive effect on accumulation (about 3-fold higher in DU-145 and 4-fold higher in HEK293), which correlates well with the higher antiproliferative activity of 3. Interestingly, despite the slightly higher accumulation in the normal cell than in the cancer cell line (about 1.4-fold), guanidinoneomycin-ruthenium conjugate (3) was more cytotoxic to cancer cells (about 1.8-fold), whereas the opposite tendency applied for neomycin-ruthenium conjugate (2). Such differences in cytotoxic activity and cellular accumulation between cancer and normal cells open the way to the creation of more selective, less toxic anticancer metallodrugs by conjugating cytotoxic metal-based complexes such as ruthenium(II) arene derivatives to guanidinoglycosides.

5-Fluorouracil in combination with cisplatin alters the microRNA expression profile in the CNE nasopharyngeal carcinoma cell line.

Chemotherapy constitutes one of the chief supplementary methods in the treatment of nasopharyngeal carcinoma (NPC). 5-Fluorouracil (5-FU) and cisplatin are classic chemotherapeutic drugs that have been widely used for NPC treatment. Although the aberrant expression of protein-coding genes has been observed after chemotherapy, the regulatory mechanisms involved remain poorly understood. MicroRNAs (miRNAs) are a newly identified class of small regulatory RNAs involved in multiple biological processes and metabolic regulation, including the initiation, progression and metastasis of human cancers. In this study, using a label-free high-throughput microRNA array technology, the stacking-hybridized universal tag (SHUT) assay, we show that 5-FU in combination with cisplatin significantly alters the global expression profile of miRNAs in CNE cells. After 48 h of treatment with a low dose [10% inhibitory concentration (IC10)] of 5-FU and/or cisplatin, numerous key miRNAs were shown to be regulated. Compared to the 431 miRNAs detected in the control cells, 184 miRNAs were significantly expressed in the 5-FU-treated cells, while 336 miRNAs were expressed in the cisplatin-treated cells and 13 miRNAs in the cells treated with the combination of both drugs. The majority of these miRNAs are associated with cancer development, progression and metastasis. This is the first time that miRNA expression profiles in the CNE cell line are shown. Our findings elucidate a potential mechanism involved in the chemotherapy of NPC and provide new clues for the treatment of NPC.

Cellular and molecular effects of the liposomal mTHPC derivative Foslipos in prostate carcinoma cells in vitro.

BACKGROUND: Meso-tetra-hydroxyphenyl-chlorine (mTHPC) is among the most powerful photosensitizers available for photodynamic therapy (PDT). However, the mechanisms leading to cell death are poorly understood. We here focused on changes at DNA and RNA levels after treatment with the liposomal mTHPC derivative Foslipos in vitro. METHODS: After determination of darktoxicity, laser conditions and uptake kinetics, PC-3 prostate carcinoma cells were subjected to PDT with Foslipos, followed by assessment of cell numbers directly (TP0) or 1h (TP1), 2h (TP2), 5h (TP5) and 24h (TP24) after illumination. Nucleic acids had been extracted for evaluation of RNA amounts and integrity as well as for estimation of abasic sites as a measure for DNA damage. Furthermore, expression changes of 84 genes related to oxidative stress were investigated by quantitative polymerase chain reaction. RESULTS: Already at TP0, the number of dead cells was significantly higher after PDT versus controls and at TP24 more than 90% of cells had been destroyed. PDT resulted in a severe damage of both RNA and DNA. Gene expression analyses revealed an impact of PDT on pathways for oxidative and metabolic stress, heat shock, proliferation and carcinogenesis, growth arrest, inflammation, DNA repair and apoptosis signaling. CONCLUSIONS: Mechanisms of Foslipos-mediated PDT comprise a combination of acute and delayed lethal effects in PC-3 cells. The latter may include death processes initiated by nucleic acid damage, activation of stress and growth arrest genes in combination with a reduced capability to adequately cope with oxidative toxicity. Our results will help to better understand molecular photodynamic effects.

Recently identified and potential targets for colon cancer treatment.

The systemic therapy for colorectal cancer has advanced from essentially a single, partially effective agent, 5-fluorouracil, to a combination of cytotoxics and antibodies offering increased survival. In addition to damage of DNA through agents, such as oxaliplatin and irinotecan, and inhibition of DNA replication, a promising approach involves modifying the control of gene expression, including epigenetic control. Modulation of invasion and metastasis should become increasingly important. Inhibition of growth-factor signaling with small-molecule drugs and antibodies can be a part of this effort. Further progress in the control of gene expression in colon cancer may be achieved with miRNAs and RNA interference if technical problems can be overcome. A number of genetic changes in colorectal cancer progression have been identified and offer targets for future therapy.

NF449 is a novel inhibitor of fibroblast growth factor receptor 3 (FGFR3) signaling active in chondrocytes and multiple myeloma cells.

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site.

Expression of glutathione S-transferase M2 in stage I/II non-small cell lung cancer and alleviation of DNA damage exposure to benzo[a]pyrene.

Glutathione S-transferases (GSTs) are a family of inducible enzymes that are important in carcinogen detoxification. GST-Mu class is showing the high activity towards most polycyclic aromatic hydrocarbon (PAH) epoxide. Our objective is to clarify the expression of GST-M2 in non-small-cell lung carcinoma (NSCLC) patients and to determine the role of GST-M2 in protecting against DNA damage. We detected changes in GST-M2 expression at mRNA levels with a panel of lung cell lines and clinical samples of malignant and paired adjacent non-malignant tissues from 50 patients with stage I or II non-small-cell lung carcinoma using real-time RT-PCR. Comet assay and gamma-H2AX were used to clarify whether DNA damaged was protected by GST-M2. Our data demonstrate that the expression of GST-M2 in tumor tissues is significantly lower than in paired adjacent non-malignant tissues (p=0.016). Loss of GST-M2 is closely associated with age, gender, T value, N value and cell differentiation (p<0.05) in early stage I/II patients. Downregulation of GST-M2 is mediated through aberrant hypermethylation in lung cancer cell lines. Protection against B[a]P-induced DNA damage by GST-M2 in lung cancer cells was detected by Comet assay and gamma-H2AX. In conclusion, DNA hypermethylation altered and reduced GST-M2 expression that resulted in susceptible to benzo[a]pyrene (B[a]P) induced DNA damage. It implies that GST-M2 reduction occurs prior to tumorigenesis.

Benzyl isothiocyanate-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of MAPK in human pancreatic cancer cells.

In our previous studies, we have shown that benzyl isothiocyanate (BITC) inhibits the growth of human pancreatic cancer cells by inducing apoptosis. In the present study, we demonstrate the activation of all the three (MAPK) family members [extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK) and P38] in response to BITC treatment. Exposure of Capan-2 cells with varying concentrations of BITC for 24 h resulted in the phosphorylation (activation) of ERK at Thr202/Tyr204, JNK at Thr183/Tyr185 and P38 at Thr180/Tyr182, leading to the induction of apoptosis. Similar MAPK activation was also observed in MiaPaCa-2 cells in response to BITC treatment. However, normal human pancreatic ductal epithelial cells did not show the activation of MAPK's and remained unaffected by BITC treatment. To confirm the role of ERK, JNK and P38 in BITC-induced G(2)/M arrest and apoptosis, Capan-2 cells were pre-treated with MAPK-specific inhibitors or MAPK8-short hairpin RNA (shRNA) prior to BITC treatment. Significant protection from BITC-induced G(2)/M arrest was observed in the cells pre-treated with MAPK kinase (MEK-1) but not JNK or P38 inhibitors. On the other hand, BITC-induced apoptosis was almost completely abrogated in the cells pre-treated with MEK-1, JNK or P38 inhibitors. Similarly, MAPK8-shRNA also offered almost complete protection against BITC-induced G(2)/M arrest and apoptosis. Furthermore, we observed that BITC treatment leads to the generation of reactive oxygen species (ROS) in Capan-2 and MiaPaCa-2 cells, which in part was orchestrated by depletion of reduced glutathione (GSH) level. Blocking ROS generation with N-acetyl-L-cysteine (NAC) significantly prevented GSH depletion and activation of ERK and JNK but not P38. Further, NAC or tiron prevented G(2)/M arrest by blocking G(2)/M regulatory proteins and completely protected the cells from BITC-induced apoptosis. Taken together, our results suggest that BITC-mediated G(2)/M arrest is mediated through ERK activation, whereas apoptosis is via ERK, JNK and P38.

The role of ATF4 stabilization and autophagy in resistance of breast cancer cells treated with Bortezomib.

The ubiquitin-proteasome system plays a key regulatory role in cellular homeostasis. The inhibition of the 26S proteasome by Bortezomib leads to the accumulation of misfolded proteins, resulting in endoplasmic reticulum stress followed by a coordinated cellular response called unfolded protein response (UPR). Endoplasmic reticulum stress is also a potent inducer of macroautophagy. Bortezomib is a selective and potent inhibitor of the 26S proteasome and is approved for the treatment of multiple myeloma. Clinical trials with Bortezomib have shown promising results for some types of cancers, but not for some others, including those of the breast. In this study, we show that Bortezomib induces the UPR and autophagy in MCF7 breast cancer cells. Surprisingly, Bortezomib did not induce phosphorylation of PERK, a key initial step of the UPR. We show that induction of autophagy by Bortezomib is dependent on the proteasomal stabilisation of ATF4 and up-regulation of LC3B by ATF4. We show that ATF4 and LC3B play a critical role in activating autophagy and protecting cells from Bortezomib-induced cell death. Our experiments also reveal that HDAC6 knockdown results in decreased LC3B protein and reduced autophagy. Our work shows that the induction of autophagy through ATF4 may be an important resistance mechanism to Bortezomib treatment in breast cancer, and targeting autophagy may represent a novel approach to sensitize breast cancers to Bortezomib.