Artesunate sensitizes ovarian cancer cells to cisplatin by downregulating RAD51

Artesunate, a semi-synthetic derivative of arteminisin originally developed for the treatment of malaria, has recently been shown to possess antitumor properties. One of the cytotoxic effects of artesunate on cancer cells is mediated by induction of oxidative stress and DNA double-strand breaks (DSBs). We report here that in addition to inducing oxidative stress and DSBs, artesunate can also downregulate RAD51 and impair DSB repair in ovarian cancer cells. We observed that the formation of RAD51 foci and homologous recombination repair (HRR) were significantly reduced in artesunate-treated cells. As a consequence, artesunate and cisplatin synergistically induced DSBs and inhibited the clonogenic formation of ovarian cancer cells. Ectopic expression of RAD51 was able to rescue the increased chemosensitivity conferred by artesunate, confirming that the chemosensitizing effect of artesuante is at least partially mediated by the downregulation of RAD51. Our results indicated that artesunatecan compromise the repair of DSBs in ovarian cancer cells, and thus could be employed as a sensitizing agent in chemotherapy.     

Mutation analysis of RAD51D in non-BRCA1/2 ovarian and breast cancer families

Background:

Recent data show that mutations in RAD51D have an aetiological role in ovarian carcinoma, yet mutations do not appear to be associated with an increased risk for breast cancer. We studied ovarian and breast cancer families having at least one woman affected by ovarian carcinoma, to assess the importance of RAD51D mutations in such families.

Methods:

The coding region of the RAD51D gene was analysed in 175 BRCA1/2-negative families with family histories of both ovarian and breast cancer ascertained from two Canadian and two Belgian institutions.

Results:

We identified one previously reported deleterious mutation, p.Arg186^* (c.556C>T), and two novel variants; missense substitution p.Cys119Arg and an intronic variant c.83-26A>G. p.Arg186^* segregated with the disease in the family and two ovarian carcinomas available for analysis showed loss of the wild-type allele, but the novel variants are likely neutral.

Conclusion:

RAD51D should be included in genetic screening of ovarian cancer families that do not have BRCA1/BRCA2 mutations. We show that mutations are more likely to be found in families with two or more ovarian cancers, or in probands with first-degree relatives with ovarian cancer, and we feel testing should be preferentially offered to affected women from such families.     

The role of RAD51 in etoposide (VP16) resistance in small cell lung cancer

Etoposide (VP16) is a potent inducer of DNA double-strand breaks (DSBs) and is efficiently used in small cell lung cancer (SCLC) therapy. However, acquired VP16 resistance remains an important barrier to effective treatment. To understand the underlying mechanisms for VP16 resistance in SCLC, we investigated DSB repair and cellular VP16 sensitivity of SCLC cells. VP16 sensitivity and RAD51, DNA-PK(cs), topoisomerase IIalpha and P-glycoprotein protein levels were determined in 17 SCLC cell lines. In order to unravel the role of RAD51 in VP16 resistance, we cloned the human RAD51 gene, transfected SCLC cells with RAD51 sense or antisense constructs and measured the VP16 resistance. Finally, we measured VP16-induced DSBs in the 17 SCLC cell lines. Two cell lines exhibited a multidrug-resistant phenotype. In the other SCLC cell lines, the cellular VP16 resistance was positively correlated with the RAD51 protein level. In addition, downregulation or overexpression of the RAD51 gene altered the VP16 sensitivity. Furthermore, the levels of the RAD51 and DNA-PK(cs) proteins were related to VP16-induced DSBs. The results suggest that repair of VP16-induced DSBs is mediated through both RAD51-dependent homologous recombination and DNA-PK(cs)-dependent nonhomologous end-joining and may be a determinant of the variation in clinical treatment effect observed in human SCLC tumors of identical histologic subtype. Finally, we propose RAD51 as a potential target to improve VP16 efficacy and predict tumor resistance in the treatment of SCLC patients.     

Enhancing the efficacy of glycolytic blockade in cancer cells via RAD51 inhibition

Targeting the early steps of the glycolysis pathway in cancers is a well-established therapeutic strategy; however, the doses required to elicit a therapeutic effect on the cancer can be toxic to the patient. Consequently, numerous preclinical and clinical studies have combined glycolytic blockade with other therapies. However, most of these other therapies do not specifically target cancer cells, and thus adversely affect normal tissue. Here we first show that a diverse number of cancer models – spontaneous, patient-derived xenografted tumor samples, and xenografted human cancer cells – can be efficiently targeted by 2-deoxy-D-Glucose (2DG), a well-known glycolytic inhibitor. Next, we tested the cancer-cell specificity of a therapeutic compound using the MEC1 cell line, a chronic lymphocytic leukemia (CLL) cell line that expresses activation induced cytidine deaminase (AID). We show that MEC1 cells, are susceptible to 4,4?-Diisothiocyano-2,2?-stilbenedisulfonic acid (DIDS), a specific RAD51 inhibitor. We then combine 2DG and DIDS, each at a lower dose and demonstrate that this combination is more efficacious than fludarabine, the current standard- of- care treatment for CLL. This suggests that the therapeutic blockade of glycolysis together with the therapeutic inhibition of RAD51-dependent homologous recombination can be a potentially beneficial combination for targeting AID positive cancer cells with minimal adverse effects on normal tissue.

Implications: Combination therapy targeting glycolysis and specific RAD51 function shows increased efficacy as compared to standard of care treatments in leukemias.     

Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations.

Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12-13 regions may collaborate in the inactivation of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination. We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12-13 regions. LOH was found in the RAD51 region in 32% of tumours, in the RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectively. Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001). The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probability, and curiously, the three patients with LOH at five regions concomitantly were under the age of 30 years. These results suggest that LOH at these regions could be related to breast cancer, and probably to a poor tumour prognosis.     

ERCC1 expression and RAD51B activity correlate with cell cycle response to platinum drug treatment not DNA repair

Background  The H69CIS200 and H69OX400 cell lines are novel models of low-level platinum-drug resistance. Resistance was not associated with increased cellular glutathione or decreased accumulation of platinum, rather the resistant cell lines have a cell cycle alteration allowing them to rapidly proliferate post drug treatment. Results  A decrease in ERCC1 protein expression and an increase in RAD51B foci activity was observed in association with the platinum induced cell cycle arrest but these changes did not correlate with resistance or altered DNA repair capacity. The H69 cells and resistant cell lines have a p53 mutation and consequently decrease expression of p21 in response to platinum drug treatment, promoting progression of the cell cycle instead of increasing p21 to maintain the arrest. Conclusion  Decreased ERCC1 protein and increased RAD51B foci may in part be mediating the maintenance of the cell cycle arrest in the sensitive cells. Resistance in the H69CIS200 and H69OX400 cells may therefore involve the regulation of ERCC1 and RAD51B independent of their roles in DNA repair. The novel mechanism of platinum resistance in the H69CIS200 and H69OX400 cells demonstrates the multifactorial nature of platinum resistance which can occur independently of alterations in DNA repair capacity and changes in ERCC1.     

替吉奥联合顺铂治疗晚期胃癌近期疗效观察（附27例）

目的：观察替吉奥联合顺铂治疗晚期胃癌的有效性和安全性。方法：采用非盲法随机对照试验。观察组15例,给予替吉奥胶囊80mg/（m2.d）,分两次口服,d1-14;顺铂75mg/（m2.d）,静脉滴注,d8。对照组12例,给予5-FU 1g/（m2.d）,持续灌注5天,顺铂100 mg/（m2.d）,d2。4周为1个疗程。治疗结束后评价疗效和生活质量状况,观察并记录不良反应。结果：观察组有效率33.3%,对照组16.7%,两组间差异无统计学意义（P〉0.05）。临床受益率、观察组（93.3%）高于对照组（58.3%）,差异具有统计学意义（P〈0.05）。治疗前后观察组与对照组Karnofsky改善情况差异无统计学意义（P〉0.05）,治疗后对照组Karnofsky评分下降较观察组明显（P〈0.05）。两组患者主要不良反应为血液学毒性、消化道反应和肝脏毒性,两组间比较差异无统计学意义（P〉0.05）。结论：替吉奥联合顺铂治疗晚期胃癌安全、有效。     

二氯乙酸钠联合顺铂对胃癌细胞的抑制作用

目的:研究丙酮酸脱氢酶激酶抑制剂二氯乙酸钠(sodium dichloroacetate,DCA)对胃癌细胞的体外抗肿瘤效应.方法:使用不同浓度的DCA分别干预处理体外培养的胃癌SGC-7901和BGC-823细胞24 h,MTT法检测胃癌细胞存活率,中位效应法观察DCA联合顺铂(cisplatin,DDP)干预的协同效应,Transwell小室实验检测胃癌细胞的侵袭性,流式细胞术双染法检测细胞的早期凋亡率.结果:DCA(20、40、60、80和100 mmol/L)以浓度依赖方式显著抑制胃癌SGC-7901和BGC-823细胞增殖,24 h的IC50值分别为60.9 mmol/L和53.8 mmol/L;较小剂量(20、40 mmol/L) DCA和DDP(5、10μmol/L)联合干预SGC-7901和BGC-823细胞的联合指数值均＜1,提示两者具有协同抑制胃癌细胞增殖的效应.与对照组相比,DCA(60、80、100 mmol/L)组SGC-7901细胞穿膜数目显著降低[(99.3±11.7)、(55.7±6.0)、(38.3±6.7) vs (182.7 ±17.3)个,均P＜0.05];同样浓度组的BGC-823穿膜细胞数目也显著降低[(88.7±8.3)、(49.0±5.7)、(42.3±6.7)vs (170.7 ±15.0)个,均P＜0.05].与对照组相比,DCA(60、80、100 mmol/L)组SGC-7901细胞的早期凋亡率显著增加[(31.7±5.2)％、(35.0±5.4)％、(37.8±6.2)％ vs (8.1±1.3)％,均P＜0.05)];BGC-823细胞的早期凋亡率也显著增加[(24.2±4.6)％、(31.9±4.2)％、(40.4±5.7)％ vs (6.6±1.4)％,均P＜0.05)].结论:DCA明显抑制体外培养胃癌细胞的增殖,并可与化疗药物产生协同效应;DCA可抑制胃癌细胞的侵袭及诱导胃癌细胞凋亡;靶向丙酮酸脱氢酶激酶有望成为未来胃癌治疗新的方法.     

替吉奥联合顺铂治疗晚期胃癌近期疗效观察(附27例)

目的:观察替吉奥联合顺铂治疗晚期胃癌的有效性和安全性。方法:采用非盲法随机对照试验。观察组15例,给予替吉奥胶囊80mg/(m2.d),分两次口服,d1-14;顺铂75mg/(m2.d),静脉滴注,d8。对照组12例,给予5-FU 1g/(m2.d),持续灌注5天,顺铂100 mg/(m2.d),d2。4周为1个疗程。治疗结束后评价疗效和生活质量状况,观察并记录不良反应。结果:观察组有效率33.3%,对照组16.7%,两组间差异无统计学意义(P>0.05)。临床受益率、观察组(93.3%)高于对照组(58.3%),差异具有统计学意义(P<0.05)。治疗前后观察组与对照组Karnofsky改善情况差异无统计学意义(P>0.05),治疗后对照组Karnofsky评分下降较观察组明显(P<0.05)。两组患者主要不良反应为血液学毒性、消化道反应和肝脏毒性,两组间比较差异无统计学意义(P>0.05)。结论:替吉奥联合顺铂治疗晚期胃癌安全、有效。     

M6A demethylase fat mass and obesity‐associated protein regulates cisplatin resistance of gastric cancer by modulating autophagy activation through ULK1

Drug resistance is an important factor for treatment failure of gastric cancer. N⁶‐methyladenosine (m⁶A) is the predominant mRNA internal modification in eukaryotes. The roles of m⁶A modification in drug resistance of gastric cancer remains unclear. In the present study, the m⁶A methylated RNA level was significantly decreased while the expression of m⁶A demethylase fat mass and obesity‐associated protein (FTO) was obviously elevated in cisplatin‐resistant (SGC‐7901/DDP) gastric cancer cells. Knockdown of FTO reversed cisplatin resistance of SGC‐7901/DDP cells both in vitro and in vivo, which was attributed to the inhibition of Unc‐51‐like kinase 1 (ULK1)‐mediated autophagy. Mechanistically, ULK1 expression was regulated in an FTO‐m⁶A‐dependent and YTHDF2‐mediated manner. Collectively, our findings indicate that the FTO/ULK1 axis exerts crucial roles in cisplatin resistance of gastric cancer.     

The variant E233G of the RAD51D gene could be a low-penetrance allele in high-risk breast cancer families without BRCA1/2 mutations

Six SNPs have been detected in the DNA repair genes RAD51C and RAD51D, not previously characterized. The novel variant E233G in RAD51D is more highly represented in high-risk, site-specific, familial breast cancer cases that are not associated with the BRCA1/2 genes, with a frequency of 5.74% (n = 174) compared to a control population (n = 567) and another subset of breast cancer patients (n = 765) with a prevalence of around 2% only (comparison to controls, OR = 2.6, 95% CI 1.12-6.03; p < 0.021). We found that the immunohistochemical profile detected in available tumors from these patients differs slightly from those described in non-BRCA1/2 tumors. Finally, the structural prediction of the putative functional consequence of this change indicates that it can diminish protein stability and structure. This suggests a role for E233G as a low-penetrance susceptibility gene in the specific subgroup of high-risk familial breast cancer cases that are not related to BRCA1/2.     

高表达磷酸丝氨酸转氨酶1抑制胃癌细胞增殖的实验研究

  目的  代谢重塑是肿瘤重要特征之一,多种关键代谢酶在肿瘤发生、发展过程中功能异常。磷酸丝氨酸转氨酶1（phosphoserine aminotransferase 1,PSAT1）催化磷酸丝氨酸生成,促进了肺腺癌、胶质瘤、乳腺癌、结肠癌等肿瘤的恶性发展。本研究分析PSAT1在胃癌中的生物功能。  方法  利用Kaplan-Meier plotter数据库分析PSAT1与胃癌患者预后的关系;利用RT-qPCR检测PSAT1在胃癌细胞株中的mRNA表达水平;构建PSAT1慢病毒高表达载体,在胃癌BGC823和NCI-N87细胞中高表达PSAT1;通过CCK-8、克隆形成和裸鼠皮下成瘤试验分析PSAT1对胃癌细胞增殖能力的影响。  结果  Kaplan-Meier plotter数据库分析显示PSAT1高表达的胃癌患者有更好的总生存期（P=1.7e-6,HR=0.52）。在胃癌KATOⅢ、AGS、SNU16、NCI-N87、MKN45、BGC823、MGC803和SGC7901细胞中,PSAT1的表达水平低于永生化的胃上皮细胞GES1。CCK8试验结果显示PSAT1高表达后可以显著抑制胃癌BGC823（120 h,P<0.000 1）和NCI-N87（96 h,P<0.000 1）细胞的体外增殖能力。克隆形成试验同样证实PSAT1可以抑制胃癌BGC823（P=0.029 6）和NCI-N87（P=0.036 5）细胞的克隆形成能力。进一步动物实验表明PSAT1抑制了BGC823细胞在裸鼠体内的增殖（n=7,P=0.009 1）。  结论  PSAT1高表达提示胃癌患者有更好的预后;PSAT1在多数胃癌细胞株中的表达低于GES1细胞;PSAT1高表达可显著抑制胃癌细胞体外、体内的增殖。     

对顺铂耐药胃癌细胞的DNA甲基化表型标志物初筛

目的:寻找和鉴定对顺铂耐药胃癌细胞的异常甲基化基因。方法:我们拟胃癌细胞和耐药细胞为研究对象,采用DNA甲基化芯片,对比分析两种细胞DNA甲基化表达谱差异,结合甲基化特异性PCR（MS-PCR）技术验证获得的异常甲基化基因。结果:利用DNA甲基化谱芯片对胃癌细胞和耐药细胞两个细胞系的基因组DNA甲基化谱进行分析,发现1 095个甲基化差异位点;并筛选出36个高甲基化,14个低甲基化修饰基因。然后通过MS-PCR方法对这50个基因的甲基化修饰在细胞水平上又进行了验证分析,最终筛选出与甲基化谱芯片结果一致的 14种基因,包括11种高甲基化和3种低甲基化修饰基因。结论:胃癌癌细胞发生顺铂耐药时,细胞基因组存在广泛的DNA甲基化修饰改变。     

沉默CXCR4的表达逆转肺癌细胞顺铂耐药

目的:探讨沉默CXCR4对肺癌细胞顺铂耐药的影响,并研究其分子作用机制。方法:利用RT-qPCR测定肺癌耐药（A549/DDP）及药物敏感细胞株（A549）中CXCR4 mRNA的表达水平;利用LipofectamineTM 2000将siRNA-CXCR4转染至肺癌耐药细胞株中,RT-qPCR及蛋白质印迹法（Western blot）验证siRNA对CXCR4基因的靶向沉默效率;利用CCK-8法检测沉默CXCR4后肺癌耐药细胞的增殖活性;利用流式细胞仪测定沉默CXCR4后肺癌耐药细胞的凋亡率;利用MTT法测定沉默CXCR4后肺癌耐药细胞对顺铂的敏感性;利用Western blot实验测定沉默CXCR4后CYP1B1蛋白的表达水平。结果:RT-qPCR实验结果显示,肺癌耐药细胞株A549/DDP中CXCR4 mRNA的表达量显著高于药物敏感细胞株（P＜0.01）;体外转染siRNA-CXCR4可明显下调A549/DDP细胞株中CXCR4的表达（P＜0.001）;CCK-8实验结果显示,沉默CXCR4的表达抑制了A549/DDP细胞的增殖活性（P＜0.01）;流式细胞仪实验结果显示,沉默CXCR4的表达促进了A549/DDP细胞凋亡（P＜0.01）;MTT实验结果显示,沉默CXCR4的表达增加了A549/DDP细胞对顺铂的敏感性（P＜0.01）;Western blot实验结果显示,沉默CXCR4后CYP1B1蛋白的表达水平显著降低（P＜0.05）。结论:沉默CXCR4的表达抑制肺癌耐药细胞增殖、促进凋亡,逆转肺癌细胞顺铂耐药,CXCR4正向调控CYP1B1的表达,可能是CXCR4逆转肺癌细胞顺铂耐药的作用机制。     

Randomised phase II study of S-1/cisplatin plus TSU-68 vs S-1/cisplatin in patients with advanced gastric cancer

Background:

This study aimed to determine whether combination S-1 plus cisplatin (CDDP) therapy, the most widely used therapy for Japanese patients with advanced gastric cancer, and the novel oral antiangiogenic agent TSU-68 could contribute to gastric cancer treatment.

Methods:

Ninety-three patients with chemotherapy-naïve unresectable or recurrent advanced gastric cancers were randomised into two groups: TSU-68 plus S-1/CDDP (group A) and S-1/CDDP (group B) groups. Both patient groups received identical S-1 and CDDP dosages. TSU-68 was orally administered for 35 consecutive days. Group B patients received S-1 orally twice daily for three consecutive weeks, followed by intravenous CDDP on day 8. The primary endpoint was progression-free survival (PFS).

Results:

Median PFS periods were 208 and 213 days in groups A and B, respectively (P=0.427). Median survival periods for groups A and B were 497.0 and 463.5 days, respectively (P=0.219). No statistically significant differences were noted for PFS, survival or the adverse event (AE) incidence rate. All AEs were expected according to previous reports for TSU-68, TS-1, and CDDP.

Conclusion:

Combination therapy involving TSU-68, S-1, and CDDP was safe and well tolerated in patients with chemotherapy-naïve unresectable or recurrent advanced gastric cancers. However, factors related to therapeutic efficacy should be investigated further.     

RAD001 shows activity against gastric cancer cells and overcomes 5-FU resistance by downregulating thymidylate synthase

We evaluated RAD001, an inhibitor of the mammalian target of rapamycin (mTOR) in human gastric cancer cell lines and determined the molecular mechanisms. RAD001 has marked growth inhibitory activity against the SNU-1 and SNU-216 cells. It inhibited phosphorylation of mTOR and S6 K, and induced G1 cell cycle arrest. Synergistic growth-inhibitory effects in combination with 5-fluorouracil (5-FU) was identified. Furthermore, RAD001 conferred sensitivity to 5-FU-resistant cell lines by downregulating thymidylate synthase (TS). In conclusion, RAD001 showed growth inhibitory activity against gastric cancer cells and acted synergistically with cytotoxic agents such as 5-FU by downregulating TS.     

同源重组修复基因RAD51可提高PTEN缺失细胞基因组稳定性

目的：研究同源重组修复基因RAD51对PTEN缺失小鼠胚胎成纤维细胞基因组稳定性的影响。方法：应用免疫荧光和中性单细胞电泳技术观察PTEN缺失后自发性和辐射诱导DNA双链断裂情况;并构建PTEN缺失细胞稳定转染RAD51的细胞系,γ射线照射后检测细胞存活率。结果：PTEN缺失导致细胞自发性和辐射诱导DNA双链断裂增多;转染RAD51后细胞存活率增高,辐射诱导的DNA双链断裂减少。结论：同源重组修复基因RAD51可以提高PTEN缺失细胞基因组稳定性。