Leucine-rich repeat-containing G protein-coupled receptor 5 marks different cancer stem cell compartments in human Caco-2 and LoVo colon cancer lines

BACKGROUND Colon cancer cell lines are widely used for research and for the screening of drugs that specifically target the stem cell compartment of colon cancers.It was reported that colon cancer carcinoma specimens contain a subset of leucine-rich repeatcontaining G protein-coupled receptor 5(LGR5)-expressing stem cells,these socalled“tumour-initiating”cells,reminiscent in their properties of the normal intestinal stem cells(ISCs),may explain the apparent heterogeneity of colon cancer cell lines.Also,colon cancer is initiated by aberrant Wnt signaling in ISCs known to express high levels of LGR5.Furthermore,in vivo reports demonstrate the clonal expansion of intestinal adenomas from a single LGR5-expressing cell.AIM To investigate whether colon cancer cell lines contain cancer stem cells and to characterize these putative cancer stem cells.METHODS A portable fluorescent reporter construct based on a conserved fragment of the LGR5 promoter was used to isolate the cell compartments expressing different levels of LGR5 in two widely used colon cancer cell lines(Caco-2 and LoVo).These cells were then characterized according to their proliferation capacity,gene expression signatures of ISC markers,and their tumorigenic properties in vivo and in vitro.RESULTS The data revealed that the LGR5 reporter can be used to identify and isolate a classical intestinal crypt stem cell-like population from the Caco-2,but not from the LoVo,cell lines,in which the cancer stem cell population is more akin to B lymphoma Moloney murine leukemia virus insertion region 1 homolog(+4 crypt)stem cells.This sub-population within Caco-2 cells exhibits an intestinal cancer stem cell gene expression signature and can both self-renew and generate differentiated LGR5 negative progeny.Our data also show that cells expressing high levels of LGR5/enhanced yellow fluorescent protein(EYFP)from this cell line exhibit tumorigenic-like properties in vivo and in vitro.In contrast,cell compartments of LoVo that are expressing high levels of LGR5/EYFP did not show these stem cell-like properties.Thus,cells that exhibit high levels of LGR5/EYFP expression represent the cancer stem cell compartment of Caco-2 colon cancer cells,but not LoVo cells.CONCLUSION Our findings highlight the presence of a spectrum of different ISC-like compartments in different colon cancer cell lines.Their existence is an important consideration for their screening applications and should be taken into account when interpreting drug screening data.We have generated a portable LGR5-reporter that serves as a valuable tool for the identification and isolation of different colon cancer stem cell populations in colon cancer lines.

Molecular analysis and anticancer properties of two identified isolates,Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2(ATCC) cell line

Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sub>50 value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and histone acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methylation-specific PCR, and the acetylated status of the histones associated with the p21wAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry.We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21 WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73,c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Human epidermal growth factor receptor 2 expression level and combined positive score can evaluate efficacy of advanced gastric cancer

BACKGROUND Although treatment options for gastric cancer(GC)continue to advance,the overall prognosis for patients with GC remains poor.At present,the predictors of treatment efficacy remain controversial except for high microsatellite instability.AIM To develop methods to identify groups of patients with GC who would benefit the most from receiving the combination of a programmed cell death protein 1(PD-1)inhibitor and chemotherapy.METHODS We acquired data from 63 patients with human epidermal growth factor receptor 2(HER2)-negative GC with a histological diagnosis of GC at the Cancer Hospital,Chinese Academy of Medical Sciences between November 2020 and October 2022.All of the patients screened received a PD-1 inhibitor combined with chemotherapy as the first-line treatment.RESULTS As of July 1,2023,the objective response rate was 61.9%,and the disease control rate was 96.8%.The median progression-free survival(mPFS)for all patients was 6.3 months.The median overall survival was not achieved.Survival analysis showed that patients with a combined positive score(CPS)≥1 exhibited an extended trend in progression-free survival(PFS)when compared to patients with a CPS of 0 after receiving a PD-1 inhibitor combined with oxaliplatin and tegafur as the first-line treatment.PFS exhibited a trend for prolongation as the expression level of HER2 increased.Based on PFS,we divided patients into two groups:A treatment group with excellent efficacy and a treatment group with poor efficacy.The mPFS of the excellent efficacy group was 8 months,with a mPFS of 9.1 months after excluding a cohort of patients who received interrupted therapy due to surgery.The mPFS was 4.5 months in patients in the group with poor efficacy who did not receive surgery.Using good/poor efficacy as the endpoint of our study,univariate analysis revealed that both CPS score(P=0.004)and HER2 expression level(P=0.015)were both factors that exerted significant influence on the efficacy of treatment the combination of a PD-1 inhibitor and chemotherapy in patients with advanced GC(AGC).Finally,multivariate analysis confirmed that CPS score was a significant influencing factor.CONCLUSION CPS score and HER2 expression both impacted the efficacy of immunotherapy combined with chemotherapy in AGC patients who were non-positive for HER2.

PADI3 induces cell cycle arrest via the Sirt2/AKT/p21 pathway and acts as a tumor suppressor gene in colon cancer

Objective:As a member of the peptidyl arginine deiminase(PAD)family,PADI3 is weakly expressed in colon cancer tissues and highly expressed in adjacent colon cancer tissues.However,the role of PADI3 in colon cancer is unclear.In this study,we investigated the function and molecular mechanism of PADI3 in colon cancer tumorigenesis.Methods:Western blot and real-time PCR were used to detect the expression levels of several genes.CCK-8,flow cytometry(FCM)and colony formation assays were used to examine cell proliferation,the cell cycle and colony formation ability.RNAsequencing analysis was used to study the molecular mechanism of PADI3 in tumorigenesis.A truncation mutation experiment was performed to determine the key functional domain of PADI3.Results:PADI3 overexpression inhibited cell proliferation and colony formation and led to G1 phase arrest in both HCT116(originating from primary colon cancer)and LoVo(originating from metastatic tumor nodules of colon cancer)cells.PADI3-expressing HCT116 cells had a lower tumor formation rate and produced smaller tumors than control cells.PADI3 significantly decreased Sirtuin2(Sirt2)and Snail expression and AKT phosphorylation and increased p21 expression,and Sirt2 overexpression partly reversed the effects induced by PADI3 overexpression.Immunocytochemistry showed that PADI3 is mainly localized in the cytoplasm.Truncation mutation experiments showed that the C-domain is the key domain involved in the antitumor activity of PADI3.Conclusions:PADI3 suppresses Snail expression and AKT phosphorylation and promotes p21 expression by downregulating Sirt2 expression in the cytoplasm,and the C-domain is the key domain for its antitumor activity.

Effects of Meloxicam on Vascular Endothelial Growth Factor and Angiopoietin-2 Expression in Colon Carcinoma Cell Line HT-29

To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 （Ang-2） protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 （CCK-8）, the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay （ELISA）. The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supernatants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.

Growth hormone releasing peptide 2 reverses anorexia associated with chemotherapy with 5-fluoruracil in colon cancer cell-bearing mice

The cancer-associated anorexia-cachexia syndrome is observed in 80% of patients with advanced-stage cancer, and is one of the major obstacles in chemo- therapy. Ghrelin is a orexigenic hormone that has been proposed to prevent anorexia. Aim of the study was to determine whether the addition of the ghrelin agonist growth hormone releasing peptide 2 (GHRP-2) to cytotoxic therapy with 5-fluoruracil (5-FU) prevents the anorexia associated with chemotherapy in cancer cachectic mice. Thirty-three BALB/c female tumourbearing mice were randomized to receive a solution containing: (a) placebo; (b) GHRP-2; (c) 5-FU; or (d) 5-FU + GHRP-2. Ten BALB/c no tumour-bearing mice received placebo solution. Food intake and survival were checked. Six hours after the drug injection the cumulative food intake was signifi cantly increased in mice treated with the combination of 5-FU + GHRP-2 versus the 5-FU alone (P = 0.0096). On day 3, the cumulative food intake of mice treated with GHRP-2,5-FU and 5-FU + GHRP-2 signifi cantly increased com- pared with naive and vehicle groups (P = 0.0007, P = 0.0038 and P = 0.0166, respectively). The median survival time was longer in 5-FU + GHRP-2 treated mice than in those with 5-FU, although it was not signifi cant (18 d versus 15.5 d, P = 0.7). For the fi rst time, we demonstrated that the addition of GHRP-2 to cytotoxic therapy with 5-FU improved appetite in tumour-bearing mice with anorexia/cachexia syndrome in early stage. These data suggest that GHRP-2 may improve the effi cacy of therapy and the quality of life of cancer patients thank to the amelioration of their nutritional state.

Targeting BCRP/ABCG2 by RNA Interference Enhances the Chemotherapy Sensitivity of Human Colon Cancer Side Population Cells

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.

Expression of Cyclooxygenase-2 in Ovarian Cancer Cell Lines

Summary： To investigate the expression of cyclooxygenase-2 （COX-2） in ovarian cancer cell lines, RT-PCR and immunocytochemistry were used to detect the expression of COX-2 in 5 ovarian cancer cell lines. The expression of COX-2 mRNA and protein was detected in all 5 cell lines. It is suggested that COX-2 is expressed in ovarian cancer cell lines, which provides a Basis for the chemoprevention of ovarian cancer.

Expression of cyclooxygenase-2 mRNA in drug-sensitive cell and drug-resistant strains of ovarian cancer cell lines

Objective： To investigate the expression of cyclooxygenase-2 （COX-2） mRNA in drug-sensitive cell and drugresistant clones of ovarian cancer cell lines. Methods： RT-PCR and immunocytochemistry were used to investigate the expression of cyclooxygenase-2 in 3 clones drug-sensitive and 5 clones drug-resistant ovarian cancer cell. Results： Strong COX-2 mRNA expressions were detected in 3 clones of drug-sensitive cell and weak expressions were detected in 5 clones of drug-resistant cell. The protein expression of COX-2 in drug-sensitive cell was strongly positive reaction in immunocytochemistry stain and there was a weak positive reaction in 5 clones of drug-resistant cell. Conclusion： The expression of COX-2 mRNA in drug-sensitive cell strains is much higher than that in drugresistant strains of ovarian cancer cell lines, providing a basis of the chemoprevention for ovarian cancer.

Quantitative expression of MMP-2 and FN in high metastatic and low metastatic cell lines of breast cancer

To analyze the relation of matrix metalloproteinase-2(MMP-2) and Fibronection (FN) mRNA expression with metastasis of breast cancer and elucidate the role of MMP-2 and FN in breast cancer metastasis.Methods The expression of MMP-2 and FN mRNA in breast cancer cell lines was detected by fluorescence-quantitative RT-PCR.The expression of MMP-2 and FN protein was detected by Western blots.Results The expression of MMP-2 and FN mRNA was down-regulated in high metastatic cell lines MDA-MB-231,MDA-MB-435,but up-regulated in low metastatic cell lines MDA-453,T47D,SK-BR-3 and non-metastatic cell line MCF-7,ZR-75-30.The protein expression of MMP-2 and FN was up-regulated in high mestastic cell lines,and down-regulated in low metastatic cell lines.Conclusion The mRNA and protein expression of MMP-2 and FN was related with breast cancer metastasis.The mRNA expression of MMP-2 and FN is feed-back regulated with protein expression.6 refs,4 figs,2 tabs.

CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

AIM:The effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological halflife of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity. METHODS:We examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by realtime reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity. RESULTS:In response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by coadministration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation. CONCLUSION:These findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC.

Ponatinib and gossypol act in synergy to suppress colorectal cancer cells by modulating apoptosis/autophagy crosstalk and inhibiting the FGF19/FGFR4 axis

Objective:To evaluate the efficacy of ponatinib plus gossypol against colorectal cancer HCT-116 and Caco-2 cells.Methods:Cells were treated with ponatinib and/or gossypol at increasing concentrations to evaluate synergistic drug interactions by combination index.Cell viability,FGF19/FGFR4,and apoptotic and autophagic cell death were studied.Results:Ponatinib(1.25-40μM)and gossypol(2.5-80μM)monotherapy inhibited HCT-116 and Caco-2 cell viability in a doseand time-dependent manner.The combination of ponatinib and gossypol at a ratio of 1 to 2 significantly decreased cell viability(P<0.05),with a>2-and>4-fold reduction in IC50,respectively,after 24 h and 48 h,as compared to the IC50 of ponatinib.Lower combined concentrations showed greater synergism(combination index<1)with a higher ponatinib dose reduction index.Moreover,ponatinib plus gossypol induced morphological changes in HCT-116and Caco-2 cells,increased beclin-1 and caspase-3,and decreased FGF19,FGFR4,Bcl-2 and p-Akt as compared to treatment with drugs alone.Conclusions:Gossypol enhances ponatinib's anticancer effects against colorectal cancer cells through antiproliferative,apoptotic,and autophagic mechanisms.This may open the way for the future use of ponatinib at lower doses with gossypol as a potentially safer targeted strategy for colorectal cancer treatment.

<i>In Vitro</i>Anticancer Activity of Plant-Derived Cannabidiol on Prostate Cancer Cell Lines

Cannabinoids, the active components of Cannabis sativa Linnaeus, have received renewed interest in recent years due to their diverse pharmacologic activities such as cell growth inhibition, anti-inflammatory effects and tumor regression, but their use in chemotherapy is limited by their psychotropic activity. To date, cannabinoids have been successfully used in the treatment of nausea and vomiting, two common side effects that accompany chemotherapy in cancer patients. Most non-THC plant cannabinoids e.g. cannabidiol and cannabigerol, seem to be devoid of psychotropic properties. However, the precise pathways through which these molecules produce an antitumor effect have not yet been fully characterized. We therefore investigated the antitumor and anti-inflammatory activities of cannabidiol (CBD) in human prostate cancer cell lines LNCaP, DU145, PC3, and assessed whether there is any advantage in using cannabis extracts enriched in cannabidiol and low in THC. Results obtained in a panel of prostate cancer cell lines clearly indicate that cannabidiol is a potent inhibitor of cancer cell growth, with significantly lower potency in non-cancer cells. The mRNA expression level of cannabinoid receptors CB1 and CB2, vascular endothelial growth factor (VEGF), PSA (prostate specific antigen) are significantly higher in human prostate cell lines. Treatment with Cannabis extract containing high CBD down regulates CB1, CB2, VEGF, PSA, pro-inflammatory cytokines/chemokine IL-6/IL-8. Our overall findings support the concept that cannabidiol, which lacks psychotropic activity, may possess anti-inflammatory property and down regulates both cannabinoid receptors, PSA, VEGF, IL-6 and IL-8. High CBD cannabis extracts are cytotoxic to androgen responsive LNCaP cells and may effectively inhibit spheroid formation in cancer stem cells. This activity may contribute to its anticancer and chemosensitizing effect against prostate cancer. Cannabidiol and other non-habit forming cannabinoids could be used as novel therapeutic agents for the treatment of prostate cancer.

Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells： an insight into the efflux-metabolism alliance

Aim The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide（CLA） enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients （Papp） of （ - ） and （ ＋ ）CLA were higher in the ab- sorptive direction than those in the secretory direction with efflux ratios（ER） of 0. 709 ± 0.411 and 0. 867± 0. 250 （ Х10^-6 -1 cm · s ）, respectively. Their bidirectional transports were significantly reduced by （75.6 ± 87.5）% af- ter treatment with verapamil （ a P-glycoprotein inhibitor） that increased the rate of metabolism by CYP3 A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of （ - ） and （ ＋ ） CLA with ERs being 2. 934 ± 1. 432 and 1. 877 ± 0. 148 （ Х 10^-6 cm/s） respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enanti- omers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following （ - ） or （ ＋ ）-isomer treatment alone. Furthermore, the uptake of （ - ）CLA was more than that of （ ＋ ）CLA in the transfected cells. Incubation with ketoeonazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexametha- sone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharma- cology of （？） CLA as a candidate drug for treatment of Alzheimer＇ s disease.

Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line

AIM To determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.METHODS Normal human colon epithelial cells(ATCC?CRL.1790?)were stimulated with either heat killed E.coli or heat killed murine cecal contents(HKC)and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production,mitochondrial autophagy and oxidant status.TNFα,IL-1βand IL-8 protein concentrations were measured within the supernatants.Fluorescent microscopy was performed to quantify the production of reactive oxygen species(ROS)using an oxidation responsive fluorogenic probe.Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria.Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells.Autophagy was detected by staining for the presence of autophagic vesicles.Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine.Mitochondrial morphology,ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software(Image J).RESULTS Exposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress.Heat killed cecal contents treatment induced a 10-12fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h(P<0.001).Heat killed E.coli stimulation resulted in a4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point(P<0.001).Both heat killed E.coli and HKC stimulated robust ROS production at 6(P<0.001),and 12 h(P<0.01).Mitochondrial morphologic abnormalities were detected at 6 and12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential,P<0.01.Microbial stimulation also induced significant autophagy at 6 and 12 h,P<0.01.Lastly,blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.CONCLUSION The findings from this study suggest that CRL.1790cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

lncRNA-CASC2对甲状腺癌细胞增殖及侵袭转移的作用

目的探讨癌易感性候选基因2(CASC2)对甲状腺癌细胞(TPC-1)增殖及侵袭转移的影响。方法收集2020年1月-2022年12月于我院行手术治疗的83例甲状腺癌患者,取甲状腺癌组织及癌旁组织进行石蜡标本制作,并采用实时荧光定量聚合酶链反应(RT-PCR)对其癌组织和癌旁组织中CASC2mRNA和miR-155-5p RNA的表达水平进行检测。将TPC-1细胞随机分为质粒组、空载组及空白组,质粒组使用脂质体转染试剂转染CASC2a;而空载组转染空载质粒,空白组不做处理。采用CCK-8法检测细胞增殖情况,Transwell侵袭实验检测细胞迁移和侵袭情况。结果癌组织中lncRNA-CASC2的表达(1.23±0.08)明显低于癌旁组织(2.16±0.11);但miR-155-5p的表达(2.18±0.16)却明显高于癌旁组织(1.46±0.10)(P<0.05),3组细胞48 h的A450(分别为0.32±0.03、0.40±0.04、0.42±0.06)、72 h(分别为0.65±0.08、0.73±0.10、0.77±0.11)及96 h(分别为0.97±0.12、1.24±0.14、1.35±0.15)间的差异均有统计学意义(P<0.05),且随着时间的增加,3组细胞的A450值均有所增大。比较3组细胞迁移和侵袭能力发现,质粒组细胞的迁移数(54.63±8.95)和侵袭数(33.58±6.63)均明显低于空载组(分别为89.37±10.04、72.37±9.72)和空白组细胞(分别为93.21±10.22、75.82±9.62)(P<0.05);而空载组和空白组细胞的迁移数和侵袭数差异无统计学意义(P>0.05)。结论LncRNA-CASC2在甲状腺癌组织中呈现低表达,而miR-150-5p在癌组织中呈高表达,两种因子均为参与甲状腺癌的发生发展的关键;且lncRNA-CASC2在细胞水平上可抑制TPC-1细胞的增殖及迁移能力,但其抑癌作用的关键点及调控机制等需进一步探索。

Upregulation of 25-hydroxyvitamin D_3-1α-hydroxylase by butyrate in Caco-2 cells

AIM: To investigate the possible involvement of 25-hydroxyvitamin D3-1α-hydroxylase [1α-25(OH)2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells.METHODS: Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 μmol/L 25(OH)2D3 or with 1μmol/L 1α-25(OH)2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25(OH)2D3 or 1α-25(OH)2D3.1α-25(OH)2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25(OH)2D3 protein.Finally, enzymatic activity was measured by ELISA.RESULTS: Both butyrate and 1α-25(OH)2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25(OH)2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25(OH)2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites.CONCLUSION: Our experimental data pr ovide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25(OH)2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25(OH)2D3 in combination with butyrate may offer a new therapeutic approach for the treatment of colon cancer.

Fermented Herbal Decoction Selectively Targeting Human Cancer Cell Line and Human Pathogenic Microorganism

Introduction: Prolonged immuno-suppressed status promised to induce internal growth of malignant cell and infectious agent, yet, only a small part of affected individuals seek medical attention or berried by commercially over-flowed fake information. Several studies have described complementary and alternative medicine as effective strategies for improving anti-infectious agent including malignant cell. The purpose of this study was to investigate the effect of a fermented herbal decoction (FHD) both in vitro and in vivo to malignant cells and microorganism by regulating leukocyte subset proportioning FHD as dietary material. Methods: In this approach of alternative study, selective anti-cancer effect by fermented decoction was tried to show first in vitro system both, cancer cell and virus strain. The fermented herbal decoction consisting of 80 sorts of herbs and fruits. The selective toxicity was set up and then for immunological factors in animal and human. The most important factor is to reduce side effect for a normal cell. Results: First, FHD was proved as safe by animal test. FHD regulated also the proportion of granulocyte and lymphocyte ratio both animal and human. In vitro culture showed selective toxicity by FHD against human melanoma and leukemia cell line but reduced toxicity was showed by normal cell line. As for the anti-virus activity, anti-virus effect was tested on the feeder layer of human fibroblast cell, after 9 days of culture. Second, FHD inhibits colon cancer growth in 3-methylholanthrene induced cancer in rat. Conclusion: The present results suggest that our fermented herbal decoction showed selective anti-cancer activities and anti-virus activities, together with the regulative effect on the immune system.

人参皂苷Rg3对结肠癌Caco-2细胞增殖和迁移的影响

目的研究人参皂苷Rg3对结肠癌Caco-2细胞的增殖和迁移能力的影响,为该药物的临床应用提供实验依据。方法MTT法检测人参皂苷Rg3处理的Caco-2细胞增殖情况,AnnexinV-PI流式细胞术检测Rg3处理的Caco-2细胞凋亡情况,实时RT-PCR和Western blot检测Bax、Bcl-2和caspase-3基因表达情况变化,ELISA检测细胞培养上清中IL-6和VEGF质量浓度,划痕实验检测细胞迁移能力。结果人参皂苷Rg3可显著降低Caco-2细胞的增殖并诱导凋亡,Rg3处理的Caco-2细胞中Bax和caspase-3表达上调,Bcl-2表达下调;Rg3可以影响细胞培养上清中IL-6和VEGF因子的分泌,Rg3处理的Caco-2细胞迁移能力。结论人参皂苷Rg3促进细胞凋亡,抑制结肠癌Caco-2细胞增殖和迁移。