HCT116 Human Colorectal Cancer Cells Research Articles

Anthocyanins, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside, inhibit immune checkpoints in human colorectal cancer cells in vitro and in silico

The objective was to assess anti-progression and stimulatory immune response effects among anthocyanins (ANC) and their metabolites on human colorectal cancer cells in vitro and in silico. Pure phenolics including delphinidin-3-O-glucoside (D3G) and its metabolites, delphinidin (DC) and gallic acid (GA), were tested alone or in combination, on HCT-116 and HT-29 human colorectal cancer cells (100–600 µg/mL). HCT-116 and HT-29 50% inhibition concentrations (µg/mL) were 396 ± 23 and 329 ± 17 for D3G; 242 ± 16 and >600 for DC; and 154 ± 5 and 81 ± 5 for GA, respectively. Using molecular docking, cyanidin-3-O-glucoside (C3G) showed the highest potential to inhibit immune checkpoints: programmed cell death protein-1 (PD-1) (−6.8 kcal/mol) and programmed death-ligand-1 (PD-L1) (−9.6 kcal/mol). C3G, D3G, DC, GA, and D3G-rich extracts decreased PD-L1 protein expression in HCT-116 cells. C3G decreased PD-L1 fluorescence intensity by 39%. ANC decreased PD-1 expression in peripheral blood mononuclear cells in monoculture by 41% and 55%, and co-culture with HCT-116 and HT-29 cells by 39% and 26% (C3G) and 50% and 51% (D3G), respectively. D3G and C3G, abundant in plant foods, showed potential for binding with and inhibiting immune checkpoints, PD-1 and PD-L1, which can activate immune response in the tumor microenvironment and induce cancer cell death.

Simulated microgravity increases polyploid giant cancer cells and nuclear localization of YAP

Physical cues are vital in determining cellular fate in cancer. In vitro 3D culture do not replicate forces present in vivo. These forces including tumor interstitial fluid pressure and matrix stiffness behave as switches in differentiation and metastasis, which are intricate features of cancer stem cells (CSCs). Gravity determines the effect of these physical factors on cell fate and functions as evident from microgravity experiments on space and ground simulations. Here, we described the role of simulation of microgravity (SMG) using rotary cell culture system (RCCS) in increasing stemness in human colorectal cancer cell HCT116. We observed distinct features of cancer stem cells including CD133/CD44 dual positive cells and migration in SMG which was not altered by autophagy induction or inhibition. 3D and SMG increased autophagy, but the flux was staggered under SMG. Increased unique giant cancer cells housing complete nuclear localization of YAP were observed in SMG. This study highlights the role of microgravity in regulating stemness in CSC and importance of physical factors in determining the same.

Bioactive Peptides Sensitize Cells to Anticancer Effects of Oxaliplatin in Human Colorectal Cancer Xenografts in Nude Mice.

Despite new agent development and short-term benefits in patients with Colorectal Cancer (CRC), metastatic CRC cure rates have not improved due to high rates of oxaliplatin resistance and toxicity. There is an urgent need for effective tools to prevent and treat CRC and reduce morbidity and mortality of CRC patients. Exploring the effects of bioactive peptides on the antitumor to CRC was of vital importance to the clinical application. This study aimed to investigate the therapeutic impact of Anticancer Bioactive Peptides (ACBP) on anticancer effect of oxaliplatin (LOHP) in human colorectal cancer xenografts models in nude mice. HCT-116 cells were cultured in vitro via CCK-8 assays and the absorbance was measured at 450 nm. Apoptosis and cell cycle were assessed by Flow Cytometry (FCM) in vitro. HCT-116 human colorectal cancer cells inoculated subcutaneously in nude mice of treatment with PBS (GG), ACBP, LOHP, ACBP+LOHP (A+L) in vivo. The quality of life was assessed by dietary amount of nude mice, the weight of nude mice, inhibition rates, tumor weight and tumor volume. Immunohistochemistry and RT-qPCR method was conducted to determine the levels of apoptosisregulating proteins/genes in transplanted tumors. ACBP induced substantial reductions in viable cell numbers and apoptosis of HCT116 cells in combined with LOHP in vitro. Compared with the control GG group, ACBP combined low dose oxaliplatin (U) group demonstrated significantly different tumor volume, the rate of apoptosis, the expression levels of Cyt-C, caspase-3,8,9 proteins and corresponding RNAs (P<0.05). The expression of pro-apoptotic proteins in the cytoplasm around the nucleus was significantly enhanced by ACBP. Short term intermittent use of ACBP alone indicted a certain inhibitory effect on tumor growth, and improve the quality of life of tumor bearing nude mice. ACBP significantly increased the anti-cancer responses of low dose oxaliplatin (L-LOHP), thus, significantly improving the quality of life of tumor-bearing nude mice.

Abstract 1870: Polyphenols from Korean artemisia annua L (pKAL) showed anti-cancer effects by multiple mechanisms on HCT116 human colorectal cancer cells

Abstract Artemisia annua L., (Gaddongsook in Korean), an annual herb, has been used for a long time in Korean folk medicine for the treatment of infectious or chronic disorder In addition, it possesses anti-inflammatory, and anti-cancer activity. However, the molecular mechanism for anti-cancer properties of Korean Artemisia annua is still unclear. Here, we investigated the anti-cancer effects of we investigated the anti-cancer effects of polyphenols from Korean Artemisia annua (pKAL) on HCT116 human colon cancer cells. pKAL induced cell death associated with the alteration of cell morphology and DNA content. Annexin V and PI staining revealed that late phase apoptosis. pKAL induced ROS generation and the cell death was recovered by NAC. Western blot revealed that pKAL significantly up-regulated the gamma-H2AX, p21, p53, p62 and cleavage of caspases-3 and PARP in HCT116 cells. pKAL induced changes in the expression of p53-dependent targets (p53, p21, Bax, Bak, and Fas). It also induced alteration of the expression of autophagy-related proteins (beclin1, p62, LC3-I, and LC3-II) posttranslational modification of proteins (Lamin A/C, HSP90, GM130) involved in the maintenance of intracellular membrane integrity. Unexpectedly, the effect of pKAL on cell death pKAL was similar in both the P53 wild-type and p53-null HCT116 colon cancer cells, although there was some difference in cell cycle arrest; in P53 wild-type cells, pKAL appeared to induce G1 arrest at lower concentration (50 μg/mL), whereas in p53-null cells, pKAL induced G2/M arrest. The cell death was regulated by JNK and p38 MAPK signaling. The cancer effects of pKAL was similar in p53-null NCI-H1299 cells. We also investigated the protein changes that is related to cell death and cell integrity with anti-body array, which revealed that pKAL induced changes in NF-Kb, PI3K, Ras, TGF-beta signaling too. In conclusion, these findings suggest that pKAL induced anticancer effects by inducing late phase apoptotic cell death at least in part through p38 MAPK and JNK pathways in HCT 1116 human colon cancer cells. It also induced alteration of cell morphology mimic to necrosis in some cells with changes of proteins involved in the maintenance of intracellular membrane integrity. These effects was p53-independent. Citation Format: Won Sup Lee, Anjugam Paramanantham, Eun Joo Jeong, Suchismita Raha, Hye Jung Kim, GonSup Kim, Sung Chul Shin. Polyphenols from Korean artemisia annua L (pKAL) showed anti-cancer effects by multiple mechanisms on HCT116 human colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1870.

Abstract 2079: Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells

Abstract Background: Mutant KRAS is a key stratification factor in determining colorectal cancer (CRC) treatment. Nearly 50% of CRC tumors have KRAS mutations and these patients are unlikely to respond to anti-EGFR therapy. 5-fluorouracil (5-FU)-based chemotherapy is the preferred treatment for CRC patients with KRAS mutations; however, response rates remain very low for patients with advanced disease. Further, a sub-set of patients treated with 5-FU-based regimens suffers from serious side effects resulting from RNA-directed effects that do not contribute to the anti-cancer activity. We are developing polymeric fluoropyrimidines, such as CF10, to treat CRC more effectively and with reduced side effects. CF10 is &amp;gt;300-fold more potent than 5-FU across the NCI 60 cell line screen with particularly strong potency to KRAS-mutant CRC cells. We investigate the role of KRAS mutation on CF10 and 5-FU response as part of an overall effort to determine if CF10-based regimens can more effectively treat CRC patients for whom 5-FU-based regimens are currently the preferred treatment option. Methods: HKe3-wtKRAS and HKe3-mtKRAS were generated from HCT-116 human CRC cells (ATCC) by transfection with the pMSCVpuro vector encoding HA-tagged mutant or wild-type KRAS. Cytotoxicity was determined using an Alamar blue assay. Thymidylate synthase (TS) inhibition was evaluated by detecting ternary complex formation by Western blot and using a TS activity assay. Topoisomerase 1 cleavage complex (Top1cc) was evaluated using a RADAR assay and by immunofluorescence. DNA double strand breaks were detected by IF for γH2AX. Results: CF10 is highly potent to CRC cells regardless of KRAS mutation status. In isogenic cell lines that differ only in KRAS, CF10 is more potent to KRAS-mutant relative to wild type cells. The improved potency of CF10 relative to 5-FU and increased sensitivity of KRAS-mutant cells is enhanced for cells grown in 3D culture. CF10 cytotoxicity is mediated by TS inhibition and Top1cc formation that induces DNA DSBs and activates apoptosis. In vivo, CF10 displays significantly improved anti-tumor activity relative to 5-FU in HCT-116 orthotopic tumors and causes minimal systemic toxicity. Conclusions. KRAS mutation confers increased sensitivity to the DNA-directed fluoropyrimidine CF10. The overall mechanism of CF10 cytotoxicity to CRC cells is similar in both KRAS-WT and KRAS-MT CRC cells and involves TS inhibition, and misincorporation of FdU into DNA followed by generation of Top1-mediated DNA DSBs. Our studies indicate FP polymers are likely to be effective in CRC regardless of KRAS-mutation. The increased sensitivity of KRAS-mutant CRC to CF10 together with the very low systemic toxicity indicates that CRC patients currently being treated with 5-FU-based therapy could more effectively be treated with CF10-based regimens. Citation Format: William H. Gmeiner, Mandira Manandhar, Amanda M. Hinds, Julija Holmes, Pierre Vidi, Toshiyuki Tsunoda, Senji Shirasawa. Enhanced potency of the polymeric fluoropyrimidine CF10 to KRAS-mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2079.

Inhibition of Karyopherin-α2 Augments Radiation-Induced Cell Death by Perturbing BRCA1-Mediated DNA Repair.

Ionizing radiation (IR) has been widely used in the treatment of cancer. Radiation-induced DNA damage triggers the DNA damage response (DDR), which can confer radioresistance and early local recurrence by activating DNA repair pathways. Since karyopherin-α2 (KPNA2), playing an important role in nucleocytoplasmic transport, was significantly increased by IR in our previous study, we aimed to determine the function of KPNA2 with regard to DDR. Exposure to radiation upregulated KPNA2 expression in human colorectal cancer HT29 and HCT116 cells and breast carcinoma MDA-MB-231 cells together with the increased expression of DNA repair protein BRCA1. The knockdown of KPNA2 effectively increased apoptotic cell death via inhibition of BRCA1 nuclear import following IR. Therefore, we propose that KPNA2 is a potential target for overcoming radioresistance via interruption to DDR.

Compound Bieshe Kang’ai inhibits proliferation and induces apoptosis in HCT116 human colorectal cancer cells

Purpose: To study the effect of Compound Bieshe Kang’ai (CBK) on proliferation and apoptosis in colorectal cancer cells.Methods: HCT116 colorectal cancer cells and FHs 74 Int intestinal cells were treated with CBK, followed by determination of cell proliferation with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Caspase-9 and caspase-3 activities as well as protein expressions of Bcl-2 and BAX, and mRNA levels of caspase-9, caspase-3, Bcl-2 and BAX in HCT116 cells were evaluated, followed by examination of the morphological alterations of HCT116 cells with Hoechst 33342 staining.Results: CBK suppressed proliferation of HCT116 cells in a concentration- and time-dependent pattern, without cytotoxicity to FHs 74 Int cells. CBK also elevated caspase-9 and caspase-3 activities, mitigated protein translation of Bcl-2 and augmented that of BAX. It also enhanced mRNA transcriptions of caspase-9, caspase-3 and BAX, but decreased that of Bcl-2 in HCT116 cells in a concentrationdependent manner, as well as induced cancer cell shrinkage, nuclear fragmentation and chromatin condensation.Conclusion: The findings highlight CBK as a promising therapeutic agent for colorectal cancers, by retarding proliferation and inducing apoptosis in cancer cells.Keywords: Apoptosis, BAX, Bcl-2, Cancer, Caspase, Compound Bieshe Kang’ai, Chromatin condensation, Nuclear fragmentation

HCT-116 colorectal cancer cells secrete chemokines which induce chemoattraction and intracellular calcium mobilization in NK92 cells.

We recently reported that pretreatment of IL-2 activated human natural killer (NK) cells with the drugs dimethyl fumarate (DMF) and monomethyl fumarate (MMF) upregulated the expression of surface chemokine receptor CCR10. Ligands for CCR10, namely CCL27 and CCL28, induced the chemotaxis of these cells. Here, we performed a bioinformatics analysis to see which chemokines might be expressed by the human HCT-116 colorectal cancer cells. We observed that, in addition to CCL27 and CCL28, HCT-116 colorectal cancer cells profoundly express CXCL16 which binds CXCR6. Consequently, NK92 cells were treated with DMF and MMF for 24h to investigate in vitro chemotaxis towards CXCL16, CCL27, and CCL28. Furthermore, supernatants collected from HCT-116 cells after 24 or 48h incubation induced the chemotaxis of NK92 cells. Similar to their effects on human IL-2-activated NK cells, MMF and DMF enhanced the expression of CCR10 and CXCR6 in NK92 cells. Neutralizing anti-CXCL16 or anti-CCL28 inhibited the chemotactic effects of 24 and 48 supernatants, whereas anti-CCL27 only inhibited the 48h supernatant activity, suggesting that 24h supernatant contains CXCL16 and CCL28, whereas HCT-116 secretes all three chemokines after 48h in vitro cultures. CXCL16, CCL27, and CCL28, as well as the supernatants collected from HCT-116, induced the mobilization of (Ca)2+ in NK92 cells. Cross-desensitization experiments confirmed the results of the chemotaxis experiments. Finally, incubation of NK92 cells with HCT-116 induced the lysis of the tumor cells. In summary, these results might have important implications in directing the anti-tumor effectors NK cells towards tumor growth sites.

Inhibition of autophagy by 3-MA promotes hypoxia-induced apoptosis in human colorectal cancer cells.

Cell autophagy reduces the sensitivity of cancer cells to therapeutic reagents in various types of human cancer. Therefore, the aim of our study was to use human colorectal cancer HCT116 cells to explore whether inhibition of autophagy by 3-Methyladenine (3-MA, an autophagy inhibitor) is able to enhance hypoxia-induced apoptosis in vitro. HCT116 cells were treated with 3-MA, hypoxia, or 3-MA plus hypoxia, and the autophagy, apoptosis and proliferation of the HCT116 cells were investigated. Western blot analysis was used to detect autophagy specificity protein microtubule-associated protein light chain 3 (LC3) expression. Effects on apoptosis were evaluated by using flow cytometry (JC-1 staining to measure mitochondrial membrane potential) and annexin V-propidium iodide (PI) staining. The results showed that the treatment of HCT116 cells in vitro with hypoxia alone increased autophagy as well as apoptosis, whereas combination treatment with 3-MA and hypoxia markedly inhibited hypoxia-induced autophagy, but increased hypoxia-induced cell apoptosis. Autophagy might play a role as a self-defense mechanism in hypoxia-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.

Abstract B199: Potent induction of immunogenic cell death by PT-112

Abstract Purpose: To characterize the ability of PT-112 to induce immunogenic cell death (ICD) in vitro and in vivo. Background: PT-112 is a novel chemical entity consisting of a platinum core conjugated to a pyrophosphate and diaminocyclohexane groups. In vitro studies demonstrated that PT-112 has both cytostatic and cytotoxic effects on human cancer cells, two effects that are seen in the absence of robust binding to nuclear DNA. Accordingly, PT-112 potency is largely unaffected by functional DNA repair pathways, suggesting that PT-112 operates with mechanisms that differ from conventional DNA-damaging chemotherapies. PT-112 is currently under phase I/II clinical development in patients with solid tumors and hematologic malignancies, both as monotherapy and in combination with a PD-L1 inhibitor. Interim reports have established encouraging tolerability and signals of single-agent anticancer activity. Previous work with human colorectal cancer HCT-116 cells demonstrated that PT-112 causes the release of ICD-relevant damage-associated molecular patterns (DAMPs). ICD is a particularly relevant form of cell death for cancer therapy as it can drive tumor-targeting immune responses of clinical relevance. Here, we tested the ability of PT-112 to induce bona fide ICD in murine breast carcinoma TSA cells by in vitro DAMP release studies and in vivo gold-standard vaccination experiments. Methods: TSA cells were exposed to PT-112, cisplatin (negative ICD control) or mitoxantrone (MTX, positive ICD control) at different concentrations for 24 and 48 hours, followed by cytofluorometric assessment of cell number, viability, cell cycle distribution and exposure of the membrane-assocaited DAMP calreticulin (CRT). In addition, culture supernatants were assayed for the release of two other DAMPs, namely ATP and HMGB1, by luminometric tests and ELISA, respectively. BALB/c mice were vaccinated with TSA cells succumbing to optimal PT-112 (or MTX and cisplatin, as positive and negative controls, respectively) concentrations or mock vaccinated (with PBS). After 14 days this was followed by a rechallenge with living TSA cells and scoring of tumor-free survival (TFS) and tumor growth. Results: In vitro, PT-112 caused a robust cytostatic and cytotoxic effect on TSA cells that was accompanied by robust CRT exposure, ATP secretion and HMGB1 release. In line with such an ICD-compatible profile, 10/10 mice vaccinated with PT-112-treated TSA cells were tumor-free after the 1st rechallenge, and also rejected a subsequent challenge performed 1 month later, suggesting the establishment of long-term immunologic anticancer memory. As expected, cisplatin-treated TSA cells failed to mediate a similar effect (TFS at D7 after rechallenge: 10%), as did the mock vaccination arm (TFS at D7: 20%). MTX-treated cells conferred partial protection (TFS at D7: 40%), and the developing tumors had significantly delayed kinetics as compared to tumors evolving in mock-vaccinated mice. Conclusions: In the TSA model system of ICD vaccination, PT-112 is superior to MTX at inducing ICD and the associated establishment of immunologic anticancer memory in vivo. These data provide a robust rationale to combining PT-112 with checkpoint inhibitors in the clinic, to achieve maximal therapeutic effects potentially coupled with long-term protection from metastatic reactivation. Citation Format: Takahiro Yamazaki, Tyler David Ames, Lorenzo Galluzzi. Potent induction of immunogenic cell death by PT-112 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B199.

RETRACTED ARTICLE: Growth inhibition and apoptosis in colorectal cancer cells induced by Vitamin D-Nanoemulsion (NVD): involvement of Wnt/β-catenin and other signal transduction pathways

BackgroundMore than the two decades, the question of whether vitamin D has a role in cancer frequency, development, and death has been premeditated in detail. Colorectal, breast, and prostate cancers have been a scrupulous spot of center, altogether, these three malignancies report for approximately 35% of cancer cases and 20% of cancer demises in the United States, and as such are a chief public health apprehension. The aim was to evaluate antitumor activity of Vitamin D-Nanoemulsion (NVD) in colorectal cancer cell lines and HCT116 xenograft model in a comprehensive approach.MethodsTwo human colorectal cancer cell lines HCT116 and HT29 (gained from College of Pharmacy, King Saud University, KSA were grown. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide protocol were performed to show the impact of NVD and β-catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines. Apoptosis/cell cycle assay was performed. Analysis was done with a FACScan (Becton–Dickinson, NJ). About 10,000 cells per sample were harvested and Histograms of DNA were analyzed with ModiFitLT software (verity Software House, ME, USA). Western blotting and RT-PCR were performed for protein and gene expression respectively in in vitro and in vivo.ResultsWe found that NVD induced cytotoxicity in colorectal cells in a dose-dependent manner and time dependent approach. Further, our data validated that NVD administration of human colorectal cancer HCT116 and HT29 cells resulted in cell growth arrest, alteration in molecules regulating cell cycle operative in the G2 phase of the cell cycle and apoptosis in a dose dependent approach. Further our results concluded that NVD administration decreases expression of β-catenin gene, AKT gene and Survivin gene and protein expression in in vitro and in vivo.ConclusionOur findings suggest that targeting β-catenin gene may encourage the alterations of cell cycle and cell cycle regulators. Wnt/β-catenin signaling pathway possibly takes part in the genesis and progression of colorectal cancer cells through regulating cell cycle and the expression of cell cycle regulators.

The Embryonic Stem Cell-Specific Transcription Factor ZFP57 Promotes Liver Metastasis of Colorectal Cancer

BackgroundThe embryonic stem cell-specific transcription factor, ZFP57, has been shown to play an important role in tumor formation. In this study, we examined if ZFP57 is involved in colorectal cancer metastasis. Materials and methodsFirst, we used colorectal cancer cell lines to perform in vivo metastatic experiments with nude mice. Next, we carried out immunohistochemical analysis of clinical specimens of colorectal cancers. ResultsIn liver metastatic experiments using human colorectal cancer HT29 and HCT116 cells, liver polymetastases occurred at high frequency in ZFP57-overexpressing HT29 and HCT116 cells, whereas both control cells only resulted in oligometastases. Next, we analyzed ZFP57 expression using clinical specimens. Liver metastasis-positive cases were more frequently associated with ZFP57 overexpression than negative cases in primary lesions of colorectal cancer, and the overexpression was particularly remarkable in tumor invasive lesions. Furthermore, ZFP57 overexpression was significantly correlated not only with liver metastasis but also with lymph node metastasis. In addition, the expression level of ZFP57 was significantly correlated with that of the metastasis-related gene NANOG. We also found that ZFP57 overexpression reduced the progression-free survival rate of patients with colorectal cancer. ConclusionsThis study demonstrated that ZFP57 plays an important role in the hematogenous metastasis of colorectal cancer, suggesting that it could be used as a novel treatment target.

Synthesis, characterization and anticancer activity of mono- and dinuclear Ni(II) and Co(II) complexes of a Schiff base derived from o-vanillin

A Schiff base ligand, 6,6′-dimethoxy-2,2′-[o-phenylenebis(nitrilomethylidyne)]diphenolato (LA), and its mononuclear and dinuclear Ni(II) and Co(II) complexes were synthesized. Structural elucidation was carried out via elemental analysis, molar conductivity, IR, NMR, UV–Vis spectroscopy, TGA and magnetic susceptibility. Single crystal X-ray diffraction of the Ni(LA) complex revealed a square planar geometry of the Ni(II) centre, with the ligand acting as a tetradentate ONNO chelate. Upon coordination to the metal centers, the ν(CN) and ν(CO) peaks shifted to lower frequencies, with appearance of new peaks assignable to ν(MN) and ν(MO) in the ranges 535–560 and 421–460 cm−1, respectively. Ni(LA) and Ni2(LA) complexes are diamagnetic, whereas the Co(LA) and Co2(LA) complexes are paramagnetic with 3 unpaired electrons each. All the complexes displayed low molar conductivities in acetonitrile indicating non-electrolytic behavior. Anticancer screening against human colorectal cancer HCT116 cell lines revealed that both dinuclear complexes were more active than their mononuclear counterparts. Ni2(LA) showed the highest potency with an IC50 value of 0.81 μM.

Clinacanthus nutans induced reactive oxygen species-dependent apoptosis and autophagy in HCT116 human colorectal cancer cells

Background: Clinacanthus nutans (Burm.f.) Lindau is a medicinal herb that is conventionally used for the treatment of skin rashes, insect bites, snake bites, diabetes, and cancer. Objective: Our study aims to investigate the apoptosis- and autophagy-inducing effects of C. nutans in HCT116 human colorectal cancer cells. Materials and Methods: Cytotoxicity of ethanol extract, hexane, ethyl acetate, and aqueous fractions of C. nutans against various cancer cell lines was determined via MTT assay. Apoptosis assays including annexin V, mito-ID, and Hoechst 33342/propidium iodide staining were carried out. The level of intracellular reactive oxidative species was determined using flow cytometry. Western blot analysis was carried out to assess the protein expression in C. nutans ethyl acetate fraction (CNEAF)-treated HCT116 cells. Results: CNEAF was found to exert the strongest cytotoxic effect against HCT116 cells (IC50 =48.81 ± 1.44 μg/mL). CNEAF-induced apoptosis was evidenced by nuclear morphological alterations, phosphatidylserine externalization, dissipation of mitochondrial membrane potential, and elevation of intracellular reactive oxygen species (ROS) level. Dissipation of mitochondrial membrane potential was attributed to the upregulation of Bax and Bak accompanied by downregulation of Bcl-2 and Bcl-xL, leading to caspase-3, -9, -8, and -10 activation. Interestingly, an upregulation of death receptor 5 was detected, suggesting involvement of intrinsic and extrinsic pathways. In addition, the occurrence of autophagy by CNEAF was supported by LC-3 accumulation and p62 degradation. The reduction of intracellular ROS level by N-acetylcysteine showed that the apoptosis and autophagy induced by CNEAF is ROS dependent. Conclusions: CNEAF induced ROS-dependent apoptosis and autophagy on HCT116 cells. Abbreviations used: CNEAF: Clinacanthus nutans ethyl acetate fraction; ROS: Reactive oxygen species; PS: Phosphatidylserine; NAC:N-Acetyl Cysteine; Bax: Bcl-2-associated X; Bcl-2: B-cell lymphoma 2; DR-5: Death receptor 5 Bak: Bcl-2-antagonist/killer 1; Bcl-xL: B-cell lymphoma-extra large.

Enhancement of oxaliplatin-induced colon cancer cell apoptosis by alantolactone, a natural product inducer of ROS.

Colon cancer is a malignant type of cancer with high prevalence and is one of the primary causes of cancer-related deaths. Oxaliplatin plays a significant role in the treatment of cancer, but the application of oxaliplatin is restricted due to its toxic side effects and drug resistance in clinical practice. Therefore, there is an urgent need for new strategies that can synergize with oxaliplatin for confronting colon cancer. Alantolactone (ALT), a natural sesquiterpene lactone, possesses antitumor properties in a number of cancer cell lines. In the present study, we investigated how ALT acts synergistically with oxaliplatin on human colorectal cancer HCT116 and RKO cells in vitro and in vivo. We observed that ALT strengthened the effect of oxaliplatin-induced growth restrain and apoptosis in HCT116 and RKO cells. It is through a mechanism concerning remarkable accumulation of intracellular reactive oxygen species (ROS) and activation of JNK and p38 MAPK signaling pathways. These changes ultimately induced apoptosis of HCT116 and RKO cells. Pretreatment of cells with the ROS reversal agent NAC significantly blocked the apoptosis induced by the combination treatment, and suppressed expression of JNK and p38 phosphorylation in HCT116 and RKO cells. In the xenograft model, the combination therapy displayed stronger antitumor activity compared with single agents. Immunohistochemistry of subsequent treatment tumors showed a significant decrease in proliferation as compared to either of the treatments alone. These results suggest that the combination treatment with ALT and oxaliplatin may become a potential therapeutic strategy for colon cancer.

Genomic and tumor biological aspects of the anticancer nicotinamide phosphoribosyltransferase inhibitor FK866 in resistant human colorectal cancer cells

Cancer cells' resistance to drugs remains an important problem affecting cancer treatment strategies. We previously studied the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor FK866's resistance mechanisms in the human colorectal cancer HCT116 cells. We established an acquired FK866-resistant cell line, HCT116RFK866. In this study, we investigated gene mutations in parental HCT116 and HCT116RFK866 cells using exome sequencing technology. The results indicated cluster genes related to NAD+ biosynthesis (including NAMPT), DNA repair, and ATP-binding cassette transporters were differentially altered in these cells. Interestingly, HCT116RFK866 cells, which are resistant to other class NAMPT inhibitors, were more sensitive to the anticancer 5-fluorouracil and cisplatin and γ-ray irradiation compared to parental HCT116 cells. This higher sensitivity appears to cause a genetic change in the identified gene clusters by resistance to the NAMPT inhibitor FK866. Collectively, these novel findings provide a better understanding of anticancer candidate NAMPT inhibitors with regard to resistance mechanisms and cancer chemotherapy strategies.

Asymmetric Synthesis and Cytotoxicity Evaluation of Right-Half Models of Antitumor Renieramycin Marine Natural Products.

A general protocol for the asymmetric synthesis of 3-N-arylmethylated right-half model compounds of renieramycins was developed, which enabled structure–activity relationship (SAR) study of several 3-N-arylmethyl derivatives. The most active compound (6a) showed significant cytotoxic activity against human prostate cancer DU145 and colorectal cancer HCT116 cell lines (IC50 = 11.9, and 12.5 nM, respectively).

Hyaluronate-Functionalized Graphene for Label-Free Electrochemical Cytosensing.

Electrochemical sensors for early tumor cell detection are currently an important area of research, as this special region directly improves the efficiency of cancer treatment. Functional graphene is a promising alternative for selective recognition and capture of target cancer cells. In our work, an effective cytosensor of hyaluronate-functionalized graphene (HG) was prepared through chemical reduction of graphene oxide. The as-prepared HG nanostructures were characterized with Fourier transform infrared spectroscopy and transmission electron microscopy coupled with cyclic voltammograms and electrochemical impedance spectroscopy, respectively. The self-assembly of HG with ethylene diamine, followed by sodium hyaluronate, enabled the fabrication of a label-free electrochemical impedance spectroscopy cytosensor with high stability and biocompatibility. Finally, the proposed cytosensor exhibited satisfying electrochemical behavior and cell-capture capacity for human colorectal cancer cells HCT-116, and also displayed a wide linear range, from 5.0 × 102 cells∙mL−1 to 5.0 × 106 cells∙mL−1, and a low detection limit of 100 cells∙mL−1 (S/N = 3) for quantification. This work paves the way for graphene applications in electrochemical cytosensing and other bioassays.

Prevention of Wogonin on Colorectal Cancer Tumorigenesis by Regulating p53 Nuclear Translocation.

The tumor suppressor protein p53 plays an important role in the development and progression of colon cancer, and the subcellular organelle localization directly affects its function. Wogonin (5,7-dihydroxy-8-methoxyflavone), a mono-flavonoid extracted from root of Scutellaria baicalensis Georgi, possesses acceptable toxicity and has been used in colorectal cancer (CRC) chemoprevention in pre-clinical trials by oncologist. However, the underlying anti-colon cancer mechanisms of wogonin are not yet fully understood. In the present study, the effect of wogonin on the initiation and development of colitis-associated cancer through p53 nuclear translocation was explored. AOM-DSS CRC animal model and human CRC HCT-116 cell model were used to evaluate the in vivo and in vitro anti-colon cancer action of wogonin. We observed that wogonin showed a dramaticlly preventive effect on colon cancer. Our results showed that wogonin caused apoptotic cell death in human CRC HCT-116 cell through increased endoplasmic reticulum (ER) stress. Meanwhile, excessive ER stress facilitated the cytoplasmic localization of p53 through increasing phosphor-p53 at S315 and S376 sites, induced caspase-dependent apoptosis and inhibited autophagy. Furthermore, we verified the chemoprevention effect and toxicity of wogonin in vivo by utilizing an AOM-DSS colon cancer animal model. We found that wogonin not only reduced tumor multiplicity, preserved colon length to normal (6.79 ± 0.34 to 7.41 ± 0.56, P < 0.05) but also didn’t induce side effects on various organs. In conclusion, these results explain the anti-tumor effect of wogonin in CRC and suggest wogonin as a potential therapeutic candidate for the therapeutic strategy in CRC treatment.

Caffeic Acid Phenethyl Ester Loaded in Microemulsions: Enhanced In Vitro Activity against Colon and Breast Cancer Cells and Possible Cellular Mechanisms

Caffeic acid phenethyl ester (CAPE) has high cytotoxicity against various cancer cells but has low water solubility and poor bioavailability. The objective of this work was to study a fully-dilutable oil-in-water microemulsion as a delivery vehicle of CAPE and the impacts on cellular uptake and expression of relevant proteins. Up to 3.30% w/w CAPE was dissolved in the microemulsion with droplets smaller than 20 nm and stable in 180 days. CAPE in the microemulsion had the significantly increased anti-proliferative activity against human HCT-116 colorectal and MCF-7 breast cancer cells than that in dimethyl sulfoxide. Dissolving CAPE in the microemulsion enhanced the cellular uptake according to fluorescence microscopy, flow cytometry, and HPLC. Subsequently, cyclin D1 was suppressed and p53 was induced, which regulated cell cycle and apoptosis, respectively, thereby enhancing anti-cancer activity. Findings from the present work suggest the potential of the studied microemulsion as a vehicle of CAPE for development of cancer intervention strategies.