Repositioning antispasmodic drug Papaverine for the treatment of chronic myeloid leukemia.

BACKGROUND: Papaverine is a benzylisoquinoline alkaloid from the plant Papaver somniferum (Opium poppy). It is approved as an antispasmodic drug by the US FDA and is also reported to have anti-cancer properties. Here, Papaverine's activity in chronic myeloid leukemia (CML) is explored using Saccharomyces cerevisiae, mammalian cancer cell lines, and in silico studies. METHODS: The sensitivity of wild-type and mutant (anti-oxidant defense, apoptosis) strains of S. cerevisiae to the drug Papaverine was tested by colony formation, spot assays, and AO/EB staining. In vitro cytotoxic effect was investigated on HCT15 (colon), A549 (lung), HeLa (cervical), and K562 (Bcr-Abl positive CML), and RAW 264.7 cell lines; cell cycle, mitochondrial membrane potential, ROS detection analyzed in K562 cells using flow cytometry and apoptotic markers, Bcr-Abl signaling pathways examined by western blotting. Molecular docking and molecular dynamics simulation of Papaverine against the target Bcr-Abl were also carried out. RESULTS: Investigation in S. cerevisiae evidenced Papaverine induces ROS-mediated apoptosis. Subsequent in vitro examination showed that CML cell line K562 was more sensitive to the drug Papaverine. Papaverine induces ROS generation, promotes apoptosis, and inhibits Bcr-Abl downstream signaling. Papaverine acts synergistically with the drug Imatinib. Furthermore, the docking and molecular dynamic simulation studies supported that Papaverine binds to the allosteric site of Bcr-Abl. CONCLUSION: The data presented here have added support to the concept of polypharmacology of existing drugs and natural compounds to interact with more than one target. This study provides a proof-of-concept for repositioning Papaverine as an anti-CML drug.

Femtosecond laser pulse assisted photoporation for drug delivery in Chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder occurs in the pluripotent hematopoietic stem cell. Currently, first-generation tyrosine kinase inhibitor (TKI) imatinib is the mainstay for the treatment of CML. Second generation TKI's like ponatinib, dasatinib, nilotinib, and bafetinib were treated against resistant CML. However, several CML patients develop resistance towards all existing inhibitors. Curcumin (Curcuma longa) a plant-derived natural compound is an effective bioactive component against various cancers including CML. Many studies have shown that curcumin induces time- and dose-dependent apoptosis in CML cells by regulating various downstream molecular regulators. Despite curcumin's selective cytotoxicity towards cancer cells, it has very poor bioavailability both in in-vitro and in-vivo conditions. In this present study, we have used femtosecond laser (fs-laser) pulses to ablate the cell membrane and standardized the conditions required for creating a cell membrane pores with less lethality. Following fs-laser pulse irradiation, K562 cells were incubated along with curcumin 30 µM for 0 h, 6 h,12 h and 24 h. Interestingly irradiated cells have shown higher sensitivity towards curcumin than non-irradiated cells. Immunoblotting studies showed higher induction levels of cleaved caspase 3 and 9 in irradiated population than non-irradiated. In summary, the results prove that irradiation by fs-laser pulses enhanced the bioavailability of curcumin and shows caspase-mediated cell death in irradiated CML cells than other populations.

Identification of natural inhibitors of Bcr-Abl for the treatment of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cells, characterized at the molecular level by the bcr/abl gene rearrangement. Even though targeting the fusion gene product Bcr-Abl protein is a successful strategy, development of drug resistance and that of drug intolerance are currently the limitations for Bcr-Abl-targeted CML therapy. With an aim to develop natural Bcr-Abl inhibitors, we performed virtual screening (VS) of ZINC natural compound database by docking with Abl kinase using Glide software. Two natural inhibitors ZINC08764498 (hit1) and ZINC12891610 (hit2) were selected by considering their high Glide docking score and critical interaction with the hinge region residue Met-318 of Abl kinase. The reactivity of the two molecules was assessed computationally by density functional theory calculations. Further, the conformational transition, hydrogen bond interactions, and the binding energies were investigated during 10-ns molecular dynamics simulation of the Abl-hit complex. When tested in vitro, hit1 compared to hit2 showed selective inhibition of cell proliferation and induction of apoptosis in Bcr-Abl-positive K-562 leukemia cells. In summary, our results demonstrate that ZINC08764498, a coumarin derivative identified through VS, is a potential natural inhibitor for the treatment of CML.

Significance of microRNA 21 in gastric cancer.

Despite promising developments of treatment, the mortality due to gastric cancer remains high and the mechanisms of gastric cancer initiation and the development also remains elusive. It has been reported that patients with positive serologic tests for H. pylori have a higher risk of the development of gastric cancer. microRNAs (miRNAs) are short non-coding RNA molecules consisting of 21-25 nucleotides (nt) in length. The miRNAs silence their cognate target genes by inhibiting mRNA translation or degrading the mRNA molecules by binding to their 3'-untranslated (UTR) regions and plays a very important role in cancer biology. Recent evidences indicate that miR-21 is overexpressed in tumour tissue, including gastric cancer and plays a vital role in tumour cell proliferation, apoptosis, invasion and angiogenesis. Elevated levels of miR-21 is associated with downregulation of tumour suppressor genes, such as programmed cell death 4 (PDCD4), tissue inhibitor of metalloproteinase 3, phosphatase and tensin homolog (PTEN), tropomyosin 1, ras homolog gene family member B, and maspin. Silencing of miR-21 through the use of a miR-21 inhibitor affected cancer cell viability, induced cell cycle arrest and increased chemosensitivity to anticancer agents indicating that miR-21 functions as an oncogene. Although an increased expression level of miR-21 has been observed in gastric cancer, studies related to the role of miR-21 in gastric cancer progression is very limited. The main thrust of this mini review is to explain the potency of miR-21 as a prognostic and/or diagnostic biomarker and as a new target for clinical therapeutic for interventions of gastric cancer progression.

Tip60-mediated acetylation activates transcription independent apoptotic activity of Abl.

BACKGROUND: The proto-oncogene, c-Abl encodes a ubiquitously expressed tyrosine kinase that critically governs the cell death response induced by genotoxic agents such as ionizing radiation and cisplatin. The catalytic function of Abl, which is essential for executing DNA damage response (DDR), is normally tightly regulated but upregulated several folds upon IR exposure due to ATM-mediated phosphorylation on S465. However, the mechanism/s leading to activation of Abl's apoptotic activity is currently unknown. RESULTS: We investigated the role of acetyl modification in regulating apoptotic activity of Abl and the results showed that DNA strand break-inducing agents, ionizing radiation and bleomycin induced Abl acetylation. Using mass spectrophotometry and site-specific acetyl antibody, we identified Abl K921, located in the DNA binding domain, and conforming to one of the lysine residue in the consensus acetylation motif (KXXK--X3-5--SGS) is acetylated following DNA damage. We further observed that the S465 phosphorylated Abl is acetyl modified during DNA damage. Signifying the modification, cells expressing the non acetylatable K921R mutant displayed attenuated apoptosis compared to wild-type in response to IR or bleomycin treatment. WT-Abl induced apoptosis irrespective of new protein synthesis. Furthermore, upon γ-irradiation K921R-Abl displayed reduced chromatin binding compared to wild type. Finally, loss of Abl K921 acetylation in Tip60-knocked down cells and co-precipitation of Abl with Tip60 in DNA damaged cells identified Tip60 as an Abl acetylase. CONCLUSION: Collective data showed that DNA damage-induced K921 Abl acetylation, mediated by Tip60, stimulates transcriptional-independent apoptotic activity and chromatin-associative property thereby defining a new regulatory mechanism governing Abl's DDR function.

The mismatch repair system modulates curcumin sensitivity through induction of DNA strand breaks and activation of G2-M checkpoint.

The highly conserved mismatch (MMR) repair system corrects postreplicative errors and modulates cellular responses to genotoxic agents. Here, we show that the MMR system strongly influences cellular sensitivity to curcumin. Compared with MMR-proficient cells, isogenically matched MMR-deficient cells displayed enhanced sensitivity to curcumin. Similarly, cells suppressed for MLH1 or MSH2 expression by RNA interference displayed increased curcumin sensitivity. Curcumin treatment generated comparable levels of reactive oxygen species and the mutagenic adduct 8-oxo-guanine in MMR-proficient and MMR-deficient cells; however, accumulation of gammaH2AX foci, a marker for DNA double-strand breaks (DSB), occurred only in MMR-positive cells in response to curcumin treatment. Additionally, MMR-positive cells showed activation of Chk1 and induction of G(2)-M cell cycle checkpoint following curcumin treatment and inhibition of Chk1 by UCN-01 abrogated Chk1 activation and heightened apoptosis in MMR-proficient cells. These results indicate that curcumin triggers the accumulation of DNA DSB and induction of a checkpoint response through a MMR-dependent mechanism. Conversely, in MMR-compromised cells, curcumin-induced DSB is significantly blunted, and as a result, cells fail to undergo cell cycle arrest, enter mitosis, and die through mitotic catastrophe. The results have potential therapeutic value, especially in the treatment of tumors with compromised MMR function.

MLH1- and ATM-dependent MAPK signaling is activated through c-Abl in response to the alkylator N-methyl-N'-nitro-N'-nitrosoguanidine.

N-Methyl-N'-nitro-N'-nitrosoguanidine (MNNG) is a DNA-methylating agent, and deficiency in mismatch repair (MMR) results in lack of sensitivity to this genotoxin (termed alkylation tolerance). A number of DNA damage response pathways are activated in a MMR-dependent manner following MNNG, and several also require ATM kinase activity. Here we show that activation of the transcription factor c-Jun is dependent upon both the MMR component MLH1 and ATM, but not ATR, in response to MNNG. In addition to c-Jun, the upstream MAPKs JNK and MKK4 are also activated in a MLH1- and ATM-dependent manner. We document that c-Jun activation is dependent on the MAPK kinase kinase MEKK1. Additionally, the tyrosine kinase c-Abl is required to activate this signaling cascade and forms a complex with MEKK1 and MLH1. This study indicates that an arm of DNA damage-activated MAPK signaling is activated in an MLH1- and ATM-dependent manner in response to MNNG and perhaps suggests that dysregulation of this signaling is responsible, in part, for alkylation tolerance.

Gadd45a contributes to p53 stabilization in response to DNA damage.

p53 is an important molecule in cellular response to DNA damage. After genotoxic stress, p53 protein stabilizes transiently and accumulates in the nucleus, where it functions as a transcription factor and upregulates multiple downstream-targeted genes, including p21(Waf1/Cip1), Gadd45a and Bax. However, regulation of p53 stabilization is complex and may mainly involve post-translational modification of p53, such as phosphorylation and acetylation. Using mouse embryonic fibroblasts (MEFs) derived from Gadd45a knockouts, we found that disruption of Gadd45a greatly abolished p53 protein stabilization following UVB treatment. Phosphorylation of p53 at Ser-15 was substantially reduced in Gadd45a-/- MEFs. In addition, p53 induction by UVB was shown to be greatly abrogated in the presence of p38 kinase inhibitor, but not c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK), suggesting that p38 protein kinase is involved in the regulation of p53 induction. Along with the findings presented above, inducible expression of Gadd45a enhanced p53 accumulation after cell exposure to UVB. Taken together, the current study demonstrates that Gadd45a, a conventional downstream gene of p53, may play a role as an upstream effector in p53 stabilization following DNA damage, and thus has defined a positive feedback signal in the activation of the p53 pathway.

GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity.

In response to DNA damage, the cell cycle checkpoint is an important biological event in maintaining genomic fidelity. Gadd45, a p53-regulated and DNA damage inducible protein, has recently been demonstrated to play a role in the G2-M checkpoint in response to DNA damage. In the current study, we further investigated the biochemical mechanism(s) involved in the GADD45-activated cell cycle G2-M arrest. Using the tetracycline-controlled system (tet-off), we established GADD45-inducible lines in HCT116 (wild-type p53) and Hela (inactivated p53 status) cells. Following inducible expression of the Gadd45 protein, cell growth was strongly suppressed in both HCT116 and Hela cells. Interestingly, HCT116 cells revealed a significant G2-M arrest but Hela cells failed to arrest at the G2-M phases, indicating that the GADD45-activated G2-M arrest requires normal p53 function. The GADD45-induced G2-M arrest was observed independent of p38 kinase activity. Importantly, induction of Gadd45 protein resulted in a reduction of nuclear cyclin B1 protein, whose nuclear localization is critical for the completion of G2-M transition. The reduced nuclear cyclin B1 levels correlated with inhibition of Cdc2/cyclin B1 kinase activity. Additionally, overexpression of cyclin B1 substantially abrogated the GADD45-induced cell growth suppression. Therefore, GADD45 inhibition of Cdc2 kinase activity through alteration of cyclin B1 subcellular localization may be an essential step in the GADD45-induced cell cycle G2-M arrest and growth suppression.

ATF3 induction following DNA damage is regulated by distinct signaling pathways and over-expression of ATF3 protein suppresses cells growth.

Mammalian cells have a remarkable diverse repertoire of response to genotoxic stress that damage DNA. Cellular responses to DNA damaging agents will initially exhibit gene induction, which is regulated by complex mechanism(s) and probably involves multiple signaling pathways. In this paper, we demonstrate that induction of ATF3 protein, a member of the ATF/CREB family of transcription factors, by ionizing radiation (IR) requires normal cellular p53 function. In contrast, induction of ATF3 after UV radiation (UV) or Methyl methanesulphonate (MMS) is independent of p53 status. Induction of ATF3 by DNA damage is rapid, transient, and through a transcriptional mechanism. The ATF3 promoter is induced by UV and MMS, but not by IR. In addition, ATF3 promoter can be activated by MEKK1, an upstream activator of the ERK and JNK kinase pathway, but not induced following p53 expression. Those results indicate that regulation of ATF3 induction after DNA damage utilizes both the p53-dependent and -independent pathways, and may also involve MAP kinase signaling pathways. Using the tetracycline-inducible system (tet-off), we have found that over-expression of ATF3 protein moderately suppresses cell growth. Interestingly, over-expression of ATF3 protein is able to slow down progression of cells from G1 to S phase, indicating that ATF3 protein might play a negative role in the control of cell cycle progression.

BRCA1 regulates GADD45 through its interactions with the OCT-1 and CAAT motifs.

BRCA1, a breast and ovarian cancer susceptibility gene, has been implicated in gene regulation. Previous studies demonstrate that BRCA1 induces GADD45, a p53-regulated and stress-inducible gene that plays an important role in cellular response to DNA damage. However, the mechanism(s) by which BRCA1 regulates GADD45 remains unclear. In this report, we have shown that BRCA1 activation of the GADD45 promoter is mediated through the OCT-1 and CAAT motifs located at the GADD45 promoter region. Site-directed mutations of both OCT-1 and CAAT motifs abrogate induction of the GADD45 promoter by BRCA1. Both OCT-1 and CAAT motifs are able to confer BRCA1 inducibility in a non-related minimal promoter. Physical associations of BRCA1 protein with transcription factors Oct-1 and NF-YA, which directly bind to the OCT-1 and CAAT motifs, are established by biotin-streptavidin pull-down and coimmunoprecipitation assays. Such protein interactions are required for interaction of BRCA1 with the GADD45 promoter because either immunodepletion of Oct-1 and NF-YA proteins or mutations in the OCT-1 and CAAT motifs disrupt BRCA1 binding to the GADD45 promoter. These findings indicate that BRCA1 can up-regulate its targeted genes through protein-protein interactions and provide a novel mechanism by which BRCA1 participates in transcriptional regulation.