Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

Metformin-loaded lecithin nanoparticles induce colorectal cancer cytotoxicity via epigenetic modulation of noncoding RNAs

Background

Colorectal cancer (CRC) is major aliment around the word, with a cumulative rate of mortality. Metformin (MT) was recently approved as anticancer drug against solid tumors, such as CRC. Resistance to MT therapy remains to be a challenging matter facing the development of possible anti-cancer strategy. To circumvent this problem, MT nano-encapsulation has been introduced to sensitize resistant cancer cells. The purpose of the current study is to explore the MT's aptitude encapsulated in lecithin (LC) and chitosan (CS) nanoparticles to inhibit CRC proliferation through modulations of long noncoding RNAs (lncRNAs), micro RNAs (miRNAs), and some biochemical markers.

Methods and results

Cytotoxic screenings of the newly synthesized MT-based regimens; MT, MT-LC NPs (NP1), MT-CS NPs (NP2), and MT-LC-CS NPs (NP3) against colorectal cancerous Caco-2 and HCT116 cell lines versus normal WI-38 cells were performed. The epigenetic mechanistic effects of these proposed regimens on lncRNAs and miRNAs were investigated. Additionally, some protein levels were assessed in CRC cells upon treatments; YKL-40, PPARγ, E-cadherin (ECN), and VEGF. We resulted that NP1 recorded the highest significant cytotoxic effect on CRC cells. HCT116 cells were more sensitive to the NP1 compared to Caco-2 cells. Intriguingly, it was suggested that NP1 tackled the CRC cells through down-regulation of the H19, HOTTIP, HULC, LINC00641, miR-200, miR‐92a, miR-21, YKL-40, PPARγ, and VEGF expressions, as well as up-regulation of the miR-944 and ECN expressions.

Conclusions

We concluded that the NP1 can potentially be cytotoxic to CRC cells in-vitro by modulating noncoding RNA.

     

Inhibition of autophagy by 3-MA potentiates purvalanol-induced apoptosis in Bax deficient HCT 116 colon cancer cells

The purine-derived analogs, roscovitine and purvalanol are selective synthetic inhibitors of cyclin-dependent kinases (CDKs) induced cell cycle arrest and lead to apoptotic cell death in various cancer cells. Although a number of studies investigated the molecular mechanism of each CDK inhibitor on apoptotic cell death mechanism with their therapeutic potential, their regulatory role on autophagy is not clarified yet. In this paper, our aim was to investigate molecular mechanism of CDK inhibitors on autophagy and apoptosis in wild type (wt) and Bax deficient HCT 116 cells. Exposure of HCT 116 wt and Bax^−/− cells to roscovitine or purvalanol for 24 h decreased cell viability in dose-dependent manner. However, Bax deficient HCT 116 cells were found more resistant against purvalanol treatment compared to wt cells. We also established that both CDK inhibitors induced apoptosis through activating mitochondria-mediated pathway in caspase-dependent manner regardless of Bax expression in HCT 116 colon cancer cells. Concomitantly, we determined that purvalanol was also effective on autophagy in HCT 116 colon cancer cells. Inhibition of autophagy by 3-MA treatment enhanced the purvalanol induced apoptotic cell death in HCT 116 Bax^−/− cells. Our results revealed that mechanistic action of each CDK inhibitor on cell death mechanism differs. While purvalanol treatment activated apoptosis and autophagy in HCT 116 cells, roscovitine was only effective on caspase-dependent apoptotic pathway. Another important difference between two CDK inhibitors, although roscovitine treatment overcame Bax-mediated drug resistance in HCT 116 cells, purvalanol did not exert same effect.     

Defective Mre11-dependent activation of Chk2 by ataxia telangiectasia mutated in colorectal carcinoma cells in response to replication-dependent DNA double strand breaks

The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.     

Synthesis, biological evaluation and molecular docking studies of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives as inhibitors of HDAC activity

In present study, a series of 3-(1,3-diphenyl-1H-pyrazol-4-yl)-N-phenylacrylamide derivatives (5a-8d) were designed, synthesized, and evaluated for HDAC inhibition and tumor cell antiproliferation. All of these compounds are reported for the first time, the chemical structures of these compounds were confirmed by means of (1)H NMR, ESI-MS and elemental analyzes. Among the compounds, compound 8c showed the most potent biological activity against HCT116 cancer cell line (IC(50) of 0.42 ± 0.02 μM for HDAC-1 and IC(50)=0.62 ± 0.02 for HCT116). Docking simulation was performed to position compound 8c into the HDAC active site to determine the probable binding model. The results of antiproliferative assay and western-blot demonstrated that compound 8c with potent inhibitory activity in tumor growth inhibition may be a potential anticancer agent against HCT116 cancer cell.     

Anticancer effect of atorvastatin nanostructured polymeric micelles based on stearyl-grafted chitosan

The purpose of this study was to develop a new therapeutic approach for atorvastatin (ATV) adopting nanostructured polymeric micelles for its controlled delivery to the cancer cells. Amphiphilic block copolymers of stearyl chitosan (SC) and sulfated stearyl chitosan (S-SC) that could self assemble to form polymeric micelles with different degree of substitution (DS) were synthesized and characterized. The synthesized chitosan derivatives were able to self assemble and form micelles encapsulating ATV with critical micellar concentrations ranging from 6.9 to 21μg/ml, drug-loading ranging from 40% to 84.1% and encapsulation efficiency ranging from 10.4% to 35%. ATV caused a significant decrease in particle size and zeta potential of both SC and S-SC micelles. Micelles encapsulating ATV exhibited a sustained release and more cytotoxic activity against MCF 7 and HCT 116 cell lines than ATV alone. The 50% cellular growth inhibition (IC50%) of the drug decreased from 10.4 to 3.7 in case of MCF 7 and from 9.4 to 3.4 in case of HCT 116 after its loading in micelles. These results indicate that SC ATV polymeric micelles can be considered as a promising system for site specific controlled delivery of ATV to tumor cells.     

Antiproliferative properties of piperidinylchalcones

Methoxylated chalcones bearing N-methylpiperidinyl substituents on ring A inhibited the growth of human tumour cell lines (MCF, HCT 116, and Jurkat) at IC50 values of <5 microM. Investigations on a representative member (12) showed that antiproliferative activity was linked to the disruption of the cell cycle at G1 and G2/M phases. The effect was concentration dependent and was evident at the approximate IC50 of 12. Down regulation of cell cycle regulatory components (CDK4, cyclin B, E2F, and phosphorylated Rb) were observed under similar conditions. Methoxylated chalcones without the piperidinyl substituent were found to exert equally potent and selective antiproliferative activity against HCT 116 tumour cells but did not interfere with cell cycle progression at their IC50 concentrations. The presence of the piperidinyl substituent in the chalcone template is proposed to lend specificity to the mechanism of antiproliferative activity, in addition to promoting a more desirable physicochemical profile.     

Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway

p53, one of the most commonly mutated genes in human cancers, is thought to be associated with cancer development. Hence, screening and identifying natural or synthetic compounds with anti-cancer activity via p53-independent pathway is one of the most challenging tasks for scientists in this field. Compound JKA97 (methoxy-1-styryl-9H-pyrid-[3,4-b]-indole) is a small molecule synthetic anti-cancer agent, with unknown mechanism(s). In this study we have demonstrated that the anti-cancer activity of JKA97 is associated with apoptotic induction via p53-independent mechanisms. We found that co-incubation of human colon cancer HCT116 cells with JKA97 inhibited HCT116 cell anchorage-independent growth in vitro and tumorigenicity in nude mice and also induced a cell apoptotic response, both in the cell culture model and in a tumorigenesis nude mouse model. Further studies showed that JKA97-induced apoptosis was dramatically impaired in Bax knock-out (Bax(-/-)) HCT116 cells, whereas the knock-out of p53 or PUMA did not show any inhibitory effects. The p53-independent apoptotic induction by JKA97 was confirmed in other colon cancer and hepatocarcinoma cell lines. In addition, our results showed an induction of Bax translocation and cytochrome c release from the mitochondria to the cytosol in HCT116 cells, demonstrating that the compound induces apoptosis through a Bax-initiated mitochondria-dependent pathway. These studies provide a molecular basis for the therapeutic application of JKA97 against human cancers with p53 mutations.     

利用TNF-α诱导建立结肠癌细胞HCTll6体外侵袭模型

该研究利用TNF—α诱导建立肿瘤细胞体外侵袭模型,为进一步的研究提供基础。利用20ng／mLTNF-α刺激结肠癌细胞HCTll67d后,使用流式细胞术检测NHCTll6细胞的CCR7和CXCR4受体表达量的变化,并利用Transwell小室检测TNF—α刺激对CCL21、SDF－1介导的细胞迁移与侵袭能力的影响。实验结果显示,20ng／mLTNF－α刺激7d后的HCTll6细胞的CCR7和CXCR4表达量均显著增加,侵袭能力也增强,且使CCL21、SDF—1介导的细胞迁移与侵袭能力显著增强。结果说明了本实验利用TNF—α诱导HCTl16细胞,成功建立了HCTl16的体外侵袭模型,为接下来的研究提供了细胞模型基础,也为进一步的药物筛选提供了基础。     

A new model for raf kinase inhibitory protein induced chemotherapeutic resistance

Therapeutic resistance remains the most challenging aspect of treating cancer. Raf kinase inhibitory protein (RKIP) emerged as a molecule capable of sensitizing cancerous cells to radio- and chemotherapy. Moreover, this small evolutionary conserved molecule, endows significant resistance to cancer therapy when its expression is reduced or lost. RKIP has been shown to inhibit the Raf-MEK-ERK, NFκB, GRK and activate the GSK3β signaling pathways. Inhibition of Raf-MEK-ERK and NFκB remains the most prominent pathways implicated in the sensitization of cells to therapeutic drugs. Our purpose was to identify a possible link between RKIP-KEAP 1-NRF2 and drug resistance. To that end, RKIP-KEAP 1 association was tested in human colorectal cancer tissues using immunohistochemistry. RKIP miRNA silencing and its inducible overexpression were employed in HEK-293 immortalized cells, HT29 and HCT116 colon cancer cell lines to further investigate our aim. We show that RKIP enhanced Kelch-like ECH-associated protein1 (KEAP 1) stability in colorectal cancer tissues and HT29 CRC cell line. RKIP silencing in immortalized HEK-293 cells (termed HEK-499) correlated significantly with KEAP 1 protein degradation and subsequent NRF2 addiction in these cells. Moreover, RKIP depletion in HEK-499, compared to control cells, bestowed resistance to supra physiological levels of H(2)O(2) and Cisplatin possibly by upregulating NF-E2-related nuclear factor 2 (NRF2) responsive genes. Similarly, we observed a direct correlation between the extent of apoptosis, after treatment with Adriamycin, and the expression levels of RKIP/KEAP 1 in HT29 but not in HCT116 CRC cells. Our data illuminate, for the first time, the NRF2-KEAP 1 pathway as a possible target for personalized therapeutic intervention in RKIP depleted cancers.     

雄黄纳米微粒在细胞水平上抑制呼吸道合胞病毒复制的初步研究

建立呼吸道合胞病毒A型(RSV-A型)感染Hep-2细胞模型,通过预防、治疗及直接灭活三种不同给药方式,观察中药雄黄对RSV-A型感染Hep-2细胞病变(CPE)的抑制作用。用高能球磨机研磨双蒸水水飞处理制备雄黄纳米微粒,应用砷钼蓝染色法测定雄黄纳米微粒浓度并在Nano Series粒度测定仪上测定其粒度。以MTT法计算药物的半数中毒剂量(TC50)。通过三种不同给药方式即预防给药、治疗给药及直接灭活给药方式进行体外实验,以利巴韦林为阳性对照药,观察雄黄纳米微粒对RSV-A型感染Hep-2细胞病变所起的作用,并对药物的量效关系进行分析。雄黄纳米微粒TC50值为0.649μg/mL。预防、治疗及直接灭活给药方式均可减轻RSV-A感染Hep-2细胞的CPE程度,其抑制RSV-A型感染Hep-2细胞病变的半数有效浓度(IC50)分别为0.20μg/mL、0.13μg/mL、0.16μg/mL,治疗指数(TI)分别为3.18、4.99和4.11,雄黄纳米微粒对RSV-A型感染Hep-2细胞病变的抑制作用存在着明显的量效关系。雄黄纳米微粒按预防、治疗及直接灭活给药方式给药时,其中治疗给药方式更有利于减轻RSV-A感染Hep-2细胞引起的病变。     

NAG-1 up-regulation mediated by EGR-1 and p53 is critical for quercetin-induced apoptosis in HCT116 colon carcinoma cells

Quercetin, a flavonoid molecule ubiquitously present in nature, has multiple effects on cancer cells, including the inhibition of cell proliferation and migration. However, the responsible molecular mechanisms are not fully understood. We found that quercetin induces the expression of NAG-1 (Non-steroidal anti-inflammatory drug activated gene-1), a TGF-β superfamily protein, during quercetin-induced apoptosis of HCT116 human colon carcinoma cells. Reporter assays using the luciferase constructs containing NAG-1 promoter region demonstrate that early growth response-1 (EGR-1) and p53 are required for quercetin-mediated activation of the NAG-1 promoter. Overexpression of NAG-1 enhanced the apoptotic effect of quercetin, but suppression of quercetin-induced NAG-1 expression by NAG-1 siRNA attenuated quercetin-induced apoptosis in HCT116 cells. Taken together, the present study demonstrates for the first time that quercetin induces apoptosis via NAG-1, providing a mechanistic basis for the apoptotic effect of quercetin in colon carcinoma cells.     

Triterpenoids from Meehania fargesii with Cytotoxic Activity1

In the investigation of Meehania fargesii, eighteen triterpenoids were isolated and identified, including a previously unknown compound with an 13,27-cycloursane skeleton, using techniques like 1D and 2D NMR, and HR-MS. Furthermore, the cytotoxicity of these compounds were evaluated against HCT116, MCF-7, and AGS cell lines using the CCK-8 method to examine their structure–activity relationship. Remarkably, compounds 13 and 16 exhibited higher cytotoxicity across all three cell lines compared to the positive drug. Western blot analysis revealed that these compounds activated apoptosis in HCT116 cells by promoting the Bax protein and inhibiting the Bcl-2 protein. This suggests that compounds 13 and 16 have potential as apoptosis-inducing agents in HCT116 cells.     

Mitochondrial dysfunction,autophagy stimulation and non-apoptotic cell death caused by nitric oxide-inducing Pt-coated Au nanoparticle in human lung carcinoma cells

BackgroundReactive oxygen species (ROS)-mediated cancer therapeutic has been at higher appreciation than those mediated by reactive nitrogen species. Cytotoxic mechanism of a novel nitric oxide (NO) inducing-Pt coated Au nanoparticle (NP) has been comparatively studied with the well-established ROS inducing Pt-based anticancer drug cisplatin in human lung A549 carcinoma cells.MethodsCytotoxicity was evaluated by MTT assay, lactate dehydrogenase (LDH) release, thiobarbituric acid substances (TBARS) and C11-Boron dipyrromethene (BODIPY). ROS (O^2·− and H₂O₂) was measured with dihydroethidium (DHE) and H₂O₂-specific sensor. Nitric oxide (NO) and mitochondrial dysfunction were evaluated respectively by NO-specific probe DAR-1 and JC-1. Autophagy was determined by lysotracker (LTR) and monodansylcadaverine (MDC) applied tandemly whereas apoptosis/necrosis by Hoechst/PI and caspase 3 activity.ResultsIC50 (concentration that inhibited cell viability by 50%) of Pt coated Au NP came to be 0.413 μM whereas IC50 of cisplatin came out to 86.5 μM in A549 cells treated for 24 h meaning NPs toxicity was over 200 times higher than cisplatin. However, no significant stimulation of intracellular ROS was observed at the IC50 of Pt coated Au NPs in A549 cells. However, markers like LDH release, TBARS, BODIPY and ROS were significantly higher due to cisplatin in comparison to Pt coated Au NP.ConclusionsPt coated Au NP caused NO-dependent mitochondrial dysfunction and autophagy. Mode of cell death due to NP was much different from ROS-inducing cisplatin.General significancePt coated Au NP offer promising opportunity in cancer therapeutic and warrants advanced study in vivo models of cancer.     

Purinergic signaling as potential target of thiamethoxam-induced neurotoxicity using silver catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>) as experimental model

Resveratrol is a polyphenolic compound in many edible foods including grapes, peanuts, and berries. Several studies have revealed the beneficial effects of resveratrol against various diseases such as heart disease, diabetes, obesity, neurological disorders, and cancer. A recent study showed that resveratrol inhibits the proliferation of HCT116 human colorectal cancer cells in three-dimensional culture (3DC) via induction of luminal apoptosis in HCT116 cell spheroids. In this study, we showed that a novel compound, caffeic acid-adducted resveratrol, has a stronger inhibitory effect on the growth of HCT116 cell spheroids in 3DC than resveratrol. It showed almost the same inhibitory efficacy as 5-fluorouracil, a conventional anticancer drug. We further showed that the resveratrol derivative did not affect the growth of HKe3 cell spheroids derived from HCT116 cells by disruption of the activating mutant KRAS gene. These results suggest that the resveratrol derivative inhibits the growth of HCT116 cell spheroids via inhibition of an oncogenic KRAS-mediated signaling pathway.     

Bak compensated for Bax in p53-null cells to release cytochrome c for the initiation of mitochondrial signaling during Withanolide D-induced apoptosis

The goal of cancer chemotherapy to induce multi-directional apoptosis as targeting a single pathway is unable to decrease all the downstream effect arises from crosstalk. Present study reports that Withanolide D (WithaD), a steroidal lactone isolated from Withania somnifera, induced cellular apoptosis in which mitochondria and p53 were intricately involved. In MOLT-3 and HCT116p53+/+ cells, WithaD induced crosstalk between intrinsic and extrinsic signaling through Bid, whereas in K562 and HCT116p53-/- cells, only intrinsic pathway was activated where Bid remain unaltered. WithaD showed pronounced activation of p53 in cancer cells. Moreover, lowered apoptogenic effect of HCT116p53-/- over HCT116p53+/+ established a strong correlation between WithaD-mediated apoptosis and p53. WithaD induced Bax and Bak upregulation in HCT116p53+/+, whereas increase only Bak expression in HCT116p53-/- cells, which was coordinated with augmented p53 expression. p53 inhibition substantially reduced Bax level and failed to inhibit Bak upregulation in HCT116p53+/+ cells confirming p53-dependent Bax and p53-independent Bak activation. Additionally, in HCT116p53+/+ cells, combined loss of Bax and Bak (HCT116Bax-Bak-) reduced WithaD-induced apoptosis and completely blocked cytochrome c release whereas single loss of Bax or Bak (HCT116Bax-Bak+/HCT116Bax+Bak-) was only marginally effective after WithaD treatment. In HCT116p53-/- cells, though Bax translocation to mitochondria was abrogated, Bak oligomerization helped the cells to release cytochrome c even before the disruption of mitochondrial membrane potential. WithaD also showed in vitro growth-inhibitory activity against an array of p53 wild type and null cancer cells and K562 xenograft in vivo. Taken together, WithaD elicited apoptosis in malignant cells through Bax/Bak dependent pathway in p53-wild type cells, whereas Bak compensated against loss of Bax in p53-null cells.     

Design, synthesis and biological evaluation of 6-pyridylmethylaminopurines as CDK inhibitors

The cyclin-dependent kinase (CDK) inhibitor seliciclib (1, CYC202) is in phase II clinical development for the treatment of cancer. Here we describe the synthesis of novel purines with greater solubility, lower metabolic clearance, and enhanced potency versus CDKs. These compounds exhibit novel selectivity profiles versus CDK isoforms. Compound αSβR-21 inhibits CDK2/cyclin E with IC(50)=30 nM, CDK7-cyclin H with IC(50)=1.3 μM, and CDK9-cyclinT with IC(50)=0.11 μM; it (CCT68127) inhibits growth of HCT116 colon cancer cells in vitro with GI(50)=0.7 μM; and shows antitumour activity when dosed p.o. at 50mg/kg to mice bearing HCT116 solid human tumour xenografts.     

Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.