In vitro cultures of circulating tumor cells:a potential tool to unravel drug sensitivity

Since taking part as leading actors in driving the metastatic process,circulating tumor cells(CTCs)have displayed a wide range of potential applications in the cancer-related research field.Besides their well-proved prognostic value,the role of CTCs in both predictive and diagnostics terms might be extremely informative about cancer properties and therefore highly helpful in the clinical decision-making process.Unfortunately,CTCs are scarcely released in the blood circulation and their counts vary a lot among different types of cancer,therefore CTC detection and consequent characterization are still highly challenging.In this context,in vitro CTC cultures could potentially offer a great opportunity to expand the number of tumor cells isolated at different stages of the disease and thus simplify the analysis of their biological and molecular features,allowing a deeper comprehension of the nature of neoplastic diseases.The aim of this review is to highlight the main attempts to establish in vitro CTC cultures from patients harboring different tumor types in order to highlight how powerful this practice could be,especially in optimizing the therapeutic strategies available in clinical practice and potentially preventing or contrasting the development of treatment resistance.

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Human hematopoietic cell K 562 , human melenoma cell LiBr and human stomach cancer cells were exposed to ultrasound (US, 1.75 W/cm\+2, 1.4, 2.16 and 2.4 MHz) in vitro in the presence or absence of hematoporphyrin (Hp, 100 μg/mL). The cell damaging effects of treatments were determined by means of the Trypan Blue dye exclusion test, MTT test and FDA test. The experimental results showed that the same cell line had different sensibilities to the US of different frequencies, and different cell line had different damage at the same acoustical radiation. The combined treatment with US and Hp enhanced greatly the cell damage, and no sensibility of insonation cells to US with Hp was observed. The cell damage tests showed that the results of MTT test corresponded well with that of Trypan Blue dye test.

Effects of Taxotere on invasive potential and multidrug resistance phenotype in pancreatic carcinoma cell line SUIT-2

INTRODUCTIONDevelopment of drug-resistance to chemotherapyand subsequent metastasis of tumor are primarilyresponsible for treatment failure and the death fromcancer. There have been many previous studies onthe relationship between expression of multidrugresistance (MDR) phenotype P-glycoprotein (P-gp)and the malignant properties of tumors, but theresults are often conflicting[1-8]. The difference intumor types or MDR phenotype induced by specificagents might account for this discrepancy. Taxotere(TXT), a member of the family of taxanes, hasantitumor activity through its effect of promotingthe polymerization of tubulin[9,10].

Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

AIM: To study the effects of aminoguanidine (AG) and two L-arginine analogues N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(omega)-nitro-L-arginine (L-NNA) on nitric oxide (NO) production induced by cytokines (TNF-alpha, IL-1 beta, and IFN-gamma) and bacterial lipopolysaccharide (LPS) mixture (CM) in the cultured rat hepatocytes, and examine their mechanisms action. METHODS: Rat hepatocytes were incubated with AG, L-NAME, L-NNA, Actinomycin D (ActD) and dexamethasone in a medium containing CM (LPS plus TNF-alpha, IL-1 beta, and IFN-gamma) for 24h. NO production in the cultured supernatant was measured with the Griess reaction. Intracellular cGMP level was detected with radioimmunoassy. RESULTS: NO production was markedly blocked by AG and L-NAME in a dose-dependent manner under inflammatory stimuli condition triggered by CM in vitro. The rate of the maximum inhibitory effects of L-NAME (38.9%) was less potent than that obtained with AG(53.7%, P 【 0.05). There was no significant difference between the inhibitory effects of AG and two L-arginine analogues on intracellular cGMP accumulation in rat cultured hepatocytes. Non-specific NOS expression inhibitor dexamethasone (DEX)and iNOS mRNA transcriptional inhibitor ActD also significantly inhibited CM-induced NO production. AG(0.1 mmol x L(-1)) and ActD (0.2 ng x L(-1)) were equipotent in decreasing NO production induced by inflammatory stimuli in vitro, and both effects were more potent than that induced by non-selectivity NOS activity inhibitor L-NAME (0.1 mmol x L(-1)) under similar stimuli conditions (P【0.01). CONCLUSION: AG is a potent selective inhibitor of inducible isoform of NOS,and the mechanism of action may be not only competitive inhibition in the substrate level, but also the gene expression level in rat hepatocytes.

Effects of“Moxibustion Serum”on Proliferation and Phenotypes of Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TIL) were cultured with “moxibustion serum”(MS), and the results were examined by flow cytometry. The results indicated that MS could enhance the proliferation of TIL,accelerate it to reach the exponential growth phase, and assist recombinant interleukin 2 (rIL-2) to enhance successively the percentage of CD3^+ positive cells, maintain the number of CD4^+ positive T cells, promote greatly the percentage of CD8^+ positive T cells among TILs, and reverse the CD4^+/CD8^+ ratio. Such cooperative effects rely on relative specificity of acupoints. It is suggested that MS is beneficial to the growth of TIL both in the aspects of proliferation and phenotypes.

Experimental study on antitumor effect of arsenic trioxide in combination with cisplatin or doxorubicin on hepatocellular carcinoma

INTRODUCTIONThe main component of a traditional Chinese drug 'Pishuang'. arsenic trioxide (As2O3), has obviously selective anti-tumor effect on human hepatocellular carcinoma (HCC)in both in vitro and in vivo studies[1-5]. Due to limited effectiveness when any anti-carcinogen is used alone and obviously increased toxicity when the dose is raised, there is no exception for As2O3. Furthermore, combined chemotherapy contributes to improve therapeutic effectiveness, disperse toxicity and surmount drug-resistance,in which the combination of traditional Chinese and modern medicine has more advantages and characteristics. As a result,we made an experimental study on anti-tumor effect of As2O3in combination with cisplantin (PDD) or doxorubicin (ADM)on HCC. to investigate the possibility of AS2O3 in combination with PDD or ADM and nature of interaction between them,and to provide experimental basis for clinical application.

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Neutron-induced apoptosis of HR8348 cells in vitro

INTRODUCTIONTo date ,the major therapy for rectal carcinoma is extensive abdomino-perineal resection[1]. Unfortunately ,after resection of rectal carcinoma ,many patients still die of blood-borne metastases ,usually in the liver or lungs ,or local prlvic recurrence[2,3],which is the major cause of morbidity and mortality in patients with rectal carcinoma .Pre-or postoperative radiotherapy can reduce the incidence of local rdcurrence[4-7].

Taxotere resistance in SUIT Taxotere resistance in pancreatic carcinoma cell line SUIT 2 and its sublines

AIM: To investigate the specific mechanisms of intrinsic and acquired resistance to taxotere (TXT) in pancreatic adenocarcinoma (PAC). METHODS: MTT assay was used to detect the sensitivity of PAC cell line SUIT-2 and its sublines (S-007, S-013, S-020, S-028 and TXT selected SUIT-2 cell line, S2/TXT) to TXT. Mdr1 (P-gp), multidrug resistance associated protein (MRP), lung resistance protein (LRP) and beta-tubulin isotype gene expressions were detected by RT-PCR. The functionality of P-gp and MRP was tested using their specific blocker verapamil (Ver) and indomethacin (IMC), respectively. The transporter activity of P-gp was also confirmed by Rhodamine 123 accumulation assay. RESULTS: S-020 and S2/TXT were found to be significantly resistant to TXT(19 and 9.5-fold to their parental cell line SUIT-2, respectively). RT-PCR demonstrated strong expression of Mdr1 in these two cell lines, but weaker expression or no expression in other cells lines. MRP and LRP expressions were found in most of these cell lines. The TXT-resistance in S2-020 and S2/TXT could be reversed almost completely by Ver, but not by IMC. Flow cytometry showed that Ver increased the accumulation of Rhodamine-123 in these two cell lines. Compared with S-020 and SUIT-2, the levels of beta-tubulin isotype II, III expressions in S-2/TXT were increased remarkably. CONCLUSION: The both intrinsic and acquired TXT-related drug resistance in these PAC cell lines is mainly mediated by P-gp, but had no relationship to MRP and LRP expressions. The increases of beta-tubulin isotype II, III might be collateral changes that occur when the SUIT-2 cells are treated with TXT.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene

AIM: To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. METHODS: Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. RESULTS: The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were 】15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P【0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P【0.05, P【0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was 】15000 and 214.5+/-31.3 micromol.L(-1), P【0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. CONCLUSION: The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

Personalized targeted therapy for esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma continues to heavily burden clinicians worldwide. Researchers have discovered the genomic landscape of esophageal squamous cell carcinoma, which holds promise for an era of personalized oncology care. One of the most pressing problems facing this issue is to improve the understanding of the newly available genomic data, and identify the driver-gene mutations, pathways, and networks. The emergence of a legion of novel targeted agents has generated much hope and hype regarding more potent treatment regimens, but the accuracy of drug selection is still arguable. Other problems, such as cancer heterogeneity, drug resistance, exceptional responders, and side effects, have to be surmounted. Evolving topics in personalized oncology, such as interpretation of genomics data, issues in targeted therapy, research approaches for targeted therapy, and future perspectives, will be discussed in this editorial.

Antisense expression of protein kinase Cα improved sensitivity to anticancerdrugs in human lung cancer LTEPa-2 cells

目的：研究蛋白激酶Ｃα（ＰＫＣα）在人肺癌ＬＴＥＰａ２细胞对一些临床抗肿瘤药物敏感性中的作用．方法：通过基因转染，免疫印迹等方法建立表达反义ＰＫＣα的人肺癌细胞模型，Ｎｏｒｔｈｅｒｎ印迹检测多药抗性基因的表达，分析了几种抗癌药物对培养细胞的ＩＣ５０．结果：表达反义ＰＫＣαＲＮＡ降低胞内ＰＫＣα水平时可抑制肺癌细胞中多药抗性基因的表达，增强肺癌细胞对抗肿瘤药物（三尖杉酯碱、卡铂、博来霉素、长春新碱、阿霉素）的敏感性．

奥希替尼在老年非小细胞肺癌患者靶向治疗中的应用效果及对T细胞水平的影响

目的 探讨奥西替尼在老年非小细胞肺癌患者靶向治疗中的效果及对免疫水平的影响。方法 回顾性选择2018年1月至2020年12月老年非小细胞肺癌患者116例研究,根据治疗方法不同分为2组,各58例。对照组采用常规放化疗治疗,观察组在对照组基础上联合奥西替尼治疗,3个月治疗后评估患者效果,比较2组总有效率、T细胞水平(CD3^(+)、CD4^(+)、CD8^(+)、CD4^(+)/CD8^(+))、肿瘤标志物水平、不良反应发生率。结果 观察组治疗3个月总有效率为44.8%高于对照组25.9%(P<0.05);2组治疗后3个月CD3^(+)、CD4^(+)、CD4^(+)/CD8^(+)水平均低于治疗前(P<0.05);CD8^(+)水平高于治疗前(P<0.05);观察组治疗后3个月CD3^(+)(58.95±4.21)%、CD4^(+)(32.59±3.11)%、CD4^(+)/CD8^(+)(1.21±0.22)高于对照组(P<0.05);CD8^(+)(26.81±3.32)%低于对照组(P<0.05);观察组干预3个月后CA125(91±8)U/ml、CYFRA21-1(1.26±0.24)μg/L及癌胚抗原(CEA)水平(34±5)μg/L均低于对照组(P<0.05);2组不良反应发生率差异无统计学意义(P>0.05)。结论 奥西替尼用于老年非小细胞肺癌患者靶向治疗中,能获得较好的总有效率,对患者T细胞水平影响较小,可降低肿瘤标志物水平,未增加不良反应发生率,值得临床推广应用。

养肺益气汤联合载药微球支气管动脉化疗在非小细胞肺癌治疗中的临床效果分析

目的:分析养肺益气汤联合载药微球支气管动脉化疗治疗非小细胞肺癌的临床效果。方法:选择2020年1月—2023年1月启东市中医院肿瘤科收治的82例非小细胞肺癌患者,根据随机数表法分为化疗组、联用组,各41例。其中化疗组采用载药微球支气管动脉化疗治疗,而联用组采用养肺益气汤联合载药微球支气管动脉化疗治疗。比较两组肿瘤标志物、中医症候积分、毒副作用发生率。结果:治疗前,两组肿瘤标志物比较,差异无统计学意义(P>0.05);治疗后,两组肿瘤标志物均低于治疗前,且联用组低于化疗组,差异有统计学意义(P<0.05)。治疗前,两组中医症候积分比较,差异无统计学意义(P>0.05);治疗后,两组中医症候积分均低于治疗前,且联用组低于化疗组,差异有统计学意义(P<0.05)。联用组毒副作用总发生率低于化疗组,差异有统计学意义(P<0.05)。结论:在针对非小细胞肺癌进行治疗时,在载药微球支气管动脉化疗基础上予以养肺益气汤治疗能够进一步控制癌症病变,缓解各项临床症状,并降低毒副作用发生的可能性。

Effect of Acanthopanax giraldii Harms Var. Hispidus Hoo polysaccharides on the human gastric cancer cell line SGC-7901 and its possible mechanism

OBJECTIVE: To study the inhibitory effect of Acanthopanax giraldii Harms Var. Hispidus Hoo polysaccharides (AGP) on SGC-7901 gastric cancer cells and its possible mechanism. METHODS: Cell doubling time analysis, colony forming assay and MTT assay were adopted to study the inhibitory effect and its characteristics. We also analyzed the amount of protein expressed by oncogenes, antioncogenes and cell factors using flow cytometric analysis. RESULTS: AGP inhibited the proliferation of SGC-7901 cells and cell colony forming ability. AGP did not inhibit the viability and function of lymphocytes of peripheral blood in healthy subjects and human embryonic tenocytes, except for the highest dosage of AGP (P

舒林酸抑制人胃腺癌细胞增殖及诱导凋亡

目的在体外对舒林酸抗人胃腺癌细胞增殖和凋亡诱导作用及其机制进行初步研究.方法采用人胃腺癌细胞株 MKN45,MKN28作为研究对象.体外药物敏感试验(MTT)检测不同浓度和作用时间的舒林酸对细胞的增殖抑制效应;分别用 Hoechst33258细胞核荧光染色、透射电镜和 DNA 琼脂糖凝胶电泳观察舒林酸诱导的细胞凋亡改变;应用 Western 斑点免疫印迹法观察经舒林酸2rnmol·L^(-1)和4mmol·L^(-1)作用24h 后细胞内环氧化酶-2(COX-2)和凋亡抑制蛋白 Bcl-2表达程度的变化.结果舒林酸对人胃腺癌细胞 MKN45,MKN28的杀伤率随着剂量的增大和作用时间的延长而增加,舒林酸对不同类型的细胞杀伤率不同.舒林酸能够诱导人胃腺癌细胞 MKN45,MKN28产生凋亡,凋亡诱导作用的强弱同样具有时间和剂量依赖性,而且对不同类型胃癌细胞的凋亡诱导效应有显著差异.经舒林酸2 mmol·L^(-1)和4mmol·L^(-1)作用24h 后,MKN45细胞内 COX-2和 Bcl-2蛋白比未经舒林酸作用的细胞表达明显减少,而 MKN28细胞内 COX-2和 Bcl-2蛋白的表达未出现明显变化.结论舒林酸在体外对人胃腺癌细胞株 MKN45和 MKN28有良好的增殖抑制作用,诱导凋亡是其抑制胃癌细胞增殖的重要作用机制.舒林酸对人胃腺癌细胞的抑制和凋亡诱导作用与其抑制细胞内环氧化酶-2,并进而抑制 Bcl-2的表达有关,并可能与胃癌细胞的分化状态有关.

姜黄素诱导肿瘤细胞凋亡的实验研究

目的:探讨姜黄素诱导HepG2细胞凋亡的作用机制。方法:应用形态学方法、AnnexinV荧光染色和流式细胞仪(FCM)检测HepG2细胞凋亡的发生,应用RT-PCR检测凋亡相关基因P53、bcl-2和Fas的变化。结果:姜黄素对HepG2细胞的生长有明显的抑制作用,并诱导肿瘤细胞发生凋亡。凋亡细胞表现为细胞固缩,核染色质碎裂;流式细胞仪检测凋亡率为11.7%,细胞停在G1和G2期。AnnexinV标记的方法检测凋亡时发现,坏死与凋亡共存。在姜黄素诱导HepG2细胞凋亡过程中,凋亡相关基因Fas转录水平比用药前增强。结论:凋亡为姜黄素抑癌的机制之一,姜黄素诱导HepG2细胞凋亡可能与P53、bcl-2和Fas基因表达有关。

肿瘤患者外周血淋巴细胞与肿瘤细胞体外化疗药敏研究

目的 :研究肿瘤患者外周血淋巴细胞 ( peripheralbloodlymphocytes ,PBL )的体外化疗药物敏感性 ,以及与肿瘤细胞化疗药敏的相关性。方法 :采用MTT法测定了 14 2例肿瘤患者PBL及其肿瘤细胞对 17种临床常用化疗药物的敏感性。结果 :神经胶质瘤患者PBL与肿瘤细胞对15种化疗药的敏感性相关性好 ,差异无统计学意义 ,P >0 0 5 ;骨肉瘤患者PBL与肿瘤细胞对 13种化疗药的敏感性相关性好 ,差异无统计学意义 ,P >0 0 5 ;乳腺癌患者PBL和肿瘤细胞对 12种化疗药物的敏感性差异有统计学意义 ,P <0 0 5或P<0 0 1。结论 :神经胶质瘤和骨肉瘤患者PBL对化疗药物的敏感性具有良好的正相关性 。