In vivo comparative therapeutic study of optimal administration of 5-fluorouracil and cisplatin using a newly established HST-1 human squamous-carcinoma cell line

Summary The efficacy and toxicity of 5-fluorouracil (5-FU) and cisplatin (CDDP) given in a sequential combination were evaluated in nude mice transplanted with HST-1, a newly established human squamous-carcinoma cell line. 5-FU and CDDP were given i.p. for 5 days and 1 day, respectively, either as single agents or in a sequential manner separated by a 24-h interval. The treatment was repeated every 30 days. Although inhibition of tumor growth was seen in all of the treated groups after two cycles, the sequence of 5-FU followed by CDDP significantly reduced the tumor burdens throughout all three courses and was more effective than the reverse sequence or either drug alone. Neither treatment-related death nor significant hematologic or nephrologic toxicities were seen, even following three cycles of therapy. Significant weight loss was observed only in mice treated with CDDP followed by 5-FU. This sequence dependence of the activity and toxicity of the 5-FU and CDDP combination should thus be incorporated into the design of a clinical trial.Supported by a Grant-in-Aid for Scientific Research (C-03670 327) from the Ministry of Education, Science and Culture, Japan     

Inhibition by 5-fluorouracil of cis-diamminedichloroplatinum(II)-induced DNA interstrand cross-link removal in a HST-1 human squamous carcinoma cell line.

To elucidate the mechanism of the synergistic cytotoxicity of 5-fluorouracil (5-FU) and cis-diamminedichloroplatinum(II) (CDDP), we studied the interaction of these agents using a human squamous carcinoma cell line (HST-1). Exposure to 5-FU for 24 h and to CDDP for 1 h produced a 50% inhibitory concentration of 1.0 micrograms/ml (7.7 microM) and 2.5 micrograms/ml (8.3 microM), respectively. The cytotoxic action of CDDP was augmented, and a greater than additive effect was observed when the cells were exposed to 5-FU (1.0 micrograms/ml; 7.7 microM) for 24 h before the CDDP treatment. This synergistic activity was maximal when the interval between 5-FU and CDDP exceeded 24 h. In contrast, the cytotoxicity of CDDP was attenuated when it preceded the exposure to 5-FU. Thymidine did not alter the 5-FU-CDDP interaction. Evaluation of the kinetics of the removal of DNA interstrand cross-links, measured by alkaline elution, showed a significant reduction of this removal in the cells exposed to 5-FU followed by CDDP with a drug-free interval of 48 h, as compared with cells exposed to CDDP alone, or to 5-FU immediately followed by CDDP, although no differences were found in the formation of DNA interstrand cross-links by CDDP among these cells. No significant differences in the accumulation of intracellular platinum were detected by atomic absorption spectrophotometry. These findings suggest that 5-FU modulates the repair of platinum-DNA adducts, thereby potentiating the antitumor activity of CDDP.     

Suppression of metastasis by tissue inhibitor of metalloproteinase-1 in a newly established human oral squamous cell carcinoma cell line

We have established a highly-metastatic cell line (designated as HNOS) and a non-metastatic cell line (designated as SAT) derived from human oral cavity squamous cell carcinoma (SCC). Both lines were transplantable in nude mice. The invasive activity through Matrigel-coated membrane of HNOS cells was also higher than that of SAT cells. mRNA of TIMP-1 was expressed in SAT cells but not in HNOS cells. Metastatic and invasive abilities were suppressed by the overexpression of TIMP-1 in HNOS cells. These results suggest that TIMP-1 may have an important role in inhibiting invasion and metastasis of human oral cavity SCC.     

AEG-1在5-氟尿嘧啶诱导的人口腔鳞状细胞癌细胞耐药中的作用

目的:探讨星形胶质细胞升高基因-1（astrocyte elevated gene-1,AEG-1）对5-氟尿嘧啶（5-FU）诱导的口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）细胞系耐药性的影响。方法:验证高（SCC15）、中（转染空质粒的对照组SCC15、CAL27）、低（CAL27）四组不同AEG-1表达量细胞模型AEG-1基因转染效果。MTT实验观察四组细胞对5-FU耐受性的变化;蛋白质印迹法和细胞划痕实验观察四组细胞在5-FU作用下caspase-3、MMP-2、MMP-9蛋白表达水平变化及细胞迁移能力的变化。结果:AEG-1基因在细胞中转染成功。MTT实验显示不同浓度5-FU作用四组细胞后,过表达组细胞的半数抑制浓度（IC50）为423.7 μg/ml,其对照组为213.5 μg/ml（P<0.05）;沉默细胞组IC50值为291.6 μg/ml,其对照组为463.1 μg/ml（P<0.05）。Western blot显示过表达组及其对照组中5-FU的浓度分别为400 μg/ml和200 μg/ml时,MMP-2、MMP-9及caspase-3的表达水平开始升高（P<0.05）;沉默组及其对照组中5-FU的浓度分别为240 μg/ml和480 μg/ml时,MMP-2、MMP-9及caspase-3的表达水平开始升高（P<0.05）。划痕实验显示加入相同浓度5-FU后过表达组细胞迁移率明显高于其对照组（P<0.05）;而沉默组细胞迁移率明显低于其对照组（P<0.05）。结论:上调/下调AEG-1表达能增加/降低OSCC细胞对5-FU的耐药性,提示OSCC细胞的耐药性可能与AEG-1表达量相关,OSCC细胞系中AEG-1的表达情况可能能间接干扰临床药物化疗的效果。     

Characterization of a newly established human pancreatic carcinoma cell line, UK Pan-1

BACKGROUND: A highly tumorigenic cell line designated as UK Pan-1 was established in a surgically removed human pancreatic adenocarcinoma and characterized as having many of the genotypic and phenotypic alterations commonly found in pancreatic tumors. METHODS: The cell line was characterized by its morphology, growth rate in monolayer culture and soft agar, tumorigenicity in nude mice, and chromosomal analysis. Furthermore, the status of p53, Ki-ras mutation and transforming growth factor (TGF)-/receptor expression were determined. The characteristics of UK Pan-1 were compared with those of other commonly used pancreatic carcinoma cell lines. RESULTS: Quiescent UK Pan-1 cells could be stimulated to proliferate in growth factor free nutrient media, indicating a growth factor independent phenotype. UK Pan- 1 cells grew in soft agar and rapidly formed tumors in nude mice. This cell line possesses a mutation at codon 12 of the c-Ki-ras-2 gene that is commonly found in pancreatic carcinoma. Fluorescence in situ hybridization showed that two alleles of p53 tumor suppressor gene were present in UK Pan-1. However, sequencing analysis revealed a mutation in one allele at exon 8, codon 273 (G to A; Arg to His). Additional growth assays indicated that the cell line was insensitive to negative growth regulation induced by exogenous TGF-beta. Molecular analysis of the TGF-beta signaling pathway showed that UK Pan-1 did not express appreciable levels of the TGF-beta receptor type I, II, or III mRNAs, but did express DPC4 mRNA. Karyotype analysis revealed an 18q21 deletion indicating a possible loss of heterozygosity for DPC4, as well as other chromosomal deletions and rearrangements. CONCLUSIONS: This study indicates that UK Pan-1 is a highly tumorigenic cell line possessing a molecularly complex pattern of mutations that may be used as a model to further the understanding of the mechanisms responsible for the development of pancreatic carcinoma.     

Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line

Background  

Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells.     

The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.     

Sex hormone response of a newly established squamous cell line derived from clinical esophageal carcinoma

Biopsy tissues from a 68-year-old Japanese man with metastases to axillary lymph nodes of a recurrent esophageal carcinoma were adapted to cell culture conditions and a continuously growing tumor cell line was developed. Immunohistochemical staining revealed that these cells contained keratinous material and the electron microscopic study revealed the presence of tonofilaments. Thus, this line, designated the KSE-1 line, was considered to have originated from metastatic squamous cell carcinoma of the esophagus. This line has a binding content of 4.2 fmol/mg protein for the estrogen receptor and 2.2 fmol/mg protein for the testosterone receptor. By measurement of cell number and thymidine incorporation, the growth rate of this line was found to be moderately responsive to these hormones, being inhibited by estrogen and enhanced by testosterone at concentration levels between 10(-13) and 10(-8) and 10(-13) and 10(-6) mg/ml, respectively.     

Production of multiple growth factors by a newly established human thyroid carcinoma cell line.

A multiple growth factor-producing tumor cell line (NIM-1) was newly established from a patient with thyroid cancer and remarkable neutrophilia. NIM-1 cells also caused severe neutrophilia in nude mice bearing tumors. NIM-1-conditioned medium (NIM-1CM) contained activities that supported not only granulocyte, macrophage and eosinophil colony formation of human bone marrow cells but also the growth of colony-stimulating factor (CSF)-dependent cell lines, NFS60-KX and TF-1. Northern blot hybridization analysis revealed the constitutive expression of granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF) and interleukin(IL)-6 mRNAs in NIM-1 cells. Enzyme-linked immunosorbent assays (ELISA) using NIM-1CM also confirmed the production of IL-1 alpha and a small amount of IL-1 beta besides G-CSF, GM-CSF and IL-6 in NIM-1 cells. In addition, unexpected production of IL-11 in NIM-1 cells was detected by northern blot hybridization analysis and by bioassay using an IL-11-dependent cell line. Therefore, NIM-1 cell line is shown to produce multiple cytokines including potentially megakaryopoietic growth factors such as GM-CSF, IL-6 and IL-11.     

Characterization of a cisplatin-resistant human ovarian carcinoma cell line expressing cross-resistance to 5-fluorouracil but collateral sensitivity to methotrexate.

In vitro exposure of the TR170 ovarian carcinoma cell line to six intermittent 24-h treatments with a 90% inhibitory concentration of cisplatin (CDDP) (0.15 micrograms/ml; 0.5 microM) resulted in a 2-fold stably resistant subline designated TR170/CP+ (B.T. Hill et al., Int. J. Cancer, 39: 219-225, 1987). Resistance to CDDP in these CP+ cells has now been associated with reduced uptake of 195mCDDP (2-fold; P less than 0.01) and decreased removal of specific Pt-DNA adducts, quantitated immunochemically, indicative of an apparent increased tolerance of CDDP-induced DNA damage. Specifically these resistant cells appeared deficient in removal of the major cis-Pt-(NH3)2d(pGpG) adduct and the difunctional cis-Pt(NH3)2d(GMP)2 lesion, showed less efficiency in removing cis-Pt(NH3)2d(pApG) adducts, but proved as proficient as the parental cell line in removing DNA-DNA interstrand cross-links. Activities of DNA polymerase-alpha and -beta were comparable in both lines, and no significant alterations in glutathione metabolism were identified. Response to acute X-irradiation was not modified in these TR170/CP+ cells, but they showed marked (10-fold) cross-resistance to 5-fluorouracil and, unusually, proved collaterally sensitive (12-fold) to methotrexate. Resistance to 5-fluorouracil was associated with significantly increased thymidylate synthase activity (P less than 0.01), but this was not reflected in altered gene expression, while increased sensitivity to methotrexate was accompanied by increased drug uptake but by unaltered activity and expression of dihydrofolate reductase. These results indicate that exposure to CDDP can result in numerous alterations, both intracellularly and at the cellular membrane, reflected in significant changes in the tumor cells' responses to the cytotoxic effects of a range of antitumor drugs. The clinical relevance of these observations remains to be established.     

Schedule-dependent interaction between paclitaxel and 5-fluorouracil in human carcinoma cell lines in vitro.

We assessed the cytotoxic interaction between paclitaxel and 5-fluorouracil administered at various schedules against four human carcinoma cell lines, A549, MCF7, PA1 and WiDr. The cells were exposed simultaneously to paclitaxel and to 5-fluorouracil for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days respectively. In another experiment, the cells were exposed simultaneously to both agents for 5 days. Cell growth inhibition was determined by MTT reduction assay. The effects of drug combinations at IC80 were analysed by the isobologram. The cytotoxic interaction of paclitaxel and 5-fluorouracil was definitely schedule dependent. Simultaneous exposure to paclitaxel and 5-fluorouracil for 24 h showed mainly subadditive effects in A549, MCF7 and WiDr cell lines, whereas it showed additive effects in PA1 cells. Sequential exposure to paclitaxel followed by 5-fluorouracil showed additive effects in all cell lines. Sequential exposure to 5-fluorouracil followed by paclitaxel showed subadditive effects in A549, MCF7 and PA1 cells. Whereas it showed additive effects in WiDr cells. These findings suggest that maximum cytotoxic effects can be obtained when paclitaxel precedes 5-fluorouracil. Interestingly, the continuous (5-day) exposure to paclitaxel and 5-fluorouracil had additive effects in A549, PA1 and WiDr cells, indicating that the prolonged simultaneous administration of these agents may circumvent the antagonistic interaction produced by short-term simultaneous administration. These findings may be useful in clinical trials of combination chemotherapy with paclitaxel and 5-fluorouracil.     

氟尿嘧啶、顺铂和表柔比星对胰腺癌细胞(Aspc-1)的相互作用

目的观察氟尿嘧啶(5-FU)、顺铂(DDP)和表柔比星(表阿霉素,E-ADM)体外对胰腺癌细胞(Aspc-1)的作用和药物联合应用时的相互作用.方法采用MTT法测定5-FU、DDP和E-ADM单用及合用时对细胞的抑制作用,并应用中效原理判定药物合用的效果.结果单用或合用时随着药物浓度的增加其对细胞的抑制作用也增加.5-FU和DDP合用时小剂量时产生协同作用(CI<1),大剂量时产生拮抗作用(CI>1).5-FU和E-ADM、DDP和E-ADM合用时产生轻度拮抗作用(CI>1).结论 5-FU和DDP合用时的相互作用与药物浓度有关,5-FU和E-ADM、DDP和E-ADM合用时在不同浓度表现为轻度拮抗作用.     

Epigenetic regulation of chemosensitivity to 5-fluorouracil and cisplatin by zebularine in oral squamous cell carcinoma

Epigenetic alterations such as histone acetylation and DNA methylation play an important role in the regulation of gene expression for cell cycles and apoptosis that may affect the chemosensitivity of cancers. Previously, we have reported that the combination of suberoylanilide hydroxamic acid (SAHA), a newly developed histone deacetylase inhibitor, with cisplatin (CDDP) possessed synergistic cytotoxicity against human oral squamous cell carcinoma (OSCC) cell line HSC-3. In this study, we used a novel DNA methyltransferase inhibitor, zebularine (Zeb), to investigate the epigenetic influence on the sensitivity of carcinoma cell lines to 5-fluorouracil (5-FU) or CDDP by evaluating apoptotic inducibility. Treatment with CDDP or 5-FU either alone or in combination with Zeb or SAHA continued for 48 or 72 h. In HSC-3 cells, Zeb had chemosensitive efficacy with CDDP, but not 5-FU, whereas SAHA showed efficacy with both CDDP and 5-FU. We showed that Zeb has strong anti-proliferative activity against HSC-3 cells, shown by decreased cellular growth and G2/M cell cycle phase accumulation. Furthermore, DNA methylation could be a regulatory mechanism for dihydropyrimidine dehydrogenase (DPD), known to be a principal factor in 5-FU resistance. CDHP (5-chloro-2,4-dihydroxypyridine), an inhibitor of DPD, had an enhancing effect on the apoptotic ability of 5-FU alone or 5-FU/Zeb combination. In conclusion, the present study suggests that low-dose (IC20) Zeb may sensitize cancer cells to CDDP, which may be an important characteristic for solid cancer treatment, and that DPD and other agents activated by Zeb in cancer cells could be an inhibitory factor in the response to apoptosis induced by 5-FU.     

Characterization of a newly established, TA-4-producing squamous carcinoma cell line derived from metastatic tongue carcinoma

A human squamous carcinoma cell line was established from the pleural effusion of a patient with recurrent squamous carcinoma of the tongue. The cell line, designated HST-I, has been passaged 82 times over a period of 4 years. The cells have been shown by light and electron microscopy to be of the squamous epithelial type. Immuno-histochemical staining was positive for keratin. When these cells were transplanted into athymic nude mice, tumors developed at the site of inoculation, which on histological examination were shown to be well-differentiated squamous carcinomas. Karyotypic analysis of cells from the cell line demonstrated an aneuploid human type with a modal chromosome number of 71, with both numerical and structural aberrations. HST-1 cells produce and secrete TA-4, a squamous-cell carcinoma-related antigen, in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation of cultured cells. Thus, the HST-1 cell line represents a new human tongue squamous carcinoma producing TA-4. This cell line appears useful for facilitating therapeutic investigations as well as biological studies on the association between cancerous growth and circulating TA-4 levels.     

Enhanced susceptibility of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5-fluorouracil

Our study was conducted to investigate whether anticancer drugs, cisplatin (CDDP) and/or 5-fluorouracil (5-FU), can modulate Fas-mediated apoptosis in oral squamous cell carcinoma (OSCC) cell lines. When OSCC cell lines, NA and HSC-4, were treated with CDDP and/or 5-FU, Fas and its mRNA expression on the plasma membrane were enhanced. An increase in caspase-3 and -8 activities was then observed by the addition of agonistic anti-Fas antibody, CH-11. Apoptosis of OSCC cells treated with anticancer drugs were significantly enhanced by CH-11, whereas untreated cells were nearly resistant to apoptosis. Moreover, the combination of CDDP and 5-FU resulted in an increasing susceptibility to apoptosis. Caspase-3 and -8 inhibitors, but not caspase-9 inhibitor, reduced Fas-mediated apoptosis enhanced by the anticancer drugs. Furthermore, OSCC cells treated with anticancer drugs exhibited decreased cellular FADD-like interleukin 1-converting enzyme-inhibitory protein (c-FLIP) levels, whereas neither the Fas-associated death domain-containing protein (FADD) nor procaspase-8 changed the expression. Moreover, antisense oligonucleotide to c-FLIP confirmed that down-regulation of c-FLIP induced sensitization to Fas-mediated apoptosis. These results suggest that CDDP and 5-FU may enhance the susceptibility to Fas-mediated apoptosis through down-regulation of c-FLIP. From these findings, a new potential strategy may be developed to improve the efficacy of anticancer drugs.     

Circular RNA ARHGAP5 inhibits cisplatin resistance in cervical squamous cell carcinoma by interacting with AUF1

Cervical squamous cell carcinoma (CSCC) is one of the leading causes of cancer death in women worldwide. Patients with advanced cervical carcinoma always have a poor prognosis once resistant to cisplatin due to the lack of effective treatment. It is urgent to investigate the molecular mechanisms of cisplatin resistance. Circular RNAs (circRNAs) are known to exert their regulatory functions in a series of malignancies. However, their effects on CSCC remain to be elucidated. Here, we found that cytoplasmic circARHGAP5, derived from second and third exons of the ARHGAP5 gene, was downregulated in cisplatin-resistant tissues compared with normal cervix tissues and untreated cervical cancer tissues. In addition, experiments from overexpression/knockdown cell lines revealed that circARHGAP5 could inhibit cisplatin-mediated cell apoptosis in CSCC cells both in vitro and in vivo. Mechanistically, circARHGAP5 interacted with AU-rich element RNA-binding protein (AUF1) directly. Overexpression of AUF1 could also inhibit cell apoptosis mediated by cisplatin. Furthermore, we detected the potential targets of AUF1 related to the apoptotic pathway and found that bcl-2-like protein 11 (BIM) was not only negatively regulated by AUF1 but positively regulated by circARHGAP5, which indicated that BIM mRNA might be degraded by AUF1 and thereby inhibited tumor cell apoptosis. Collectively, our data indicated that circARHGAP5 directly bound to AUF1 and prevented AUF1 from interacting with BIM mRNA, thereby playing a pivotal role in cisplatin resistance in CSCC. Our study provides insights into overcoming cancer resistance to cisplatin treatment.     

Downregulation of midkine induces cisplatin resistance in human oral squamous cell carcinoma

The presence of drug-resistant cancer cells has been associated with poor clinical outcomes. Cisplatin is one of the most effective chemotherapeutic agents commonly used for several malignancies including oral squamous cell carcinoma (OSCC). Although cisplatin resistance is a major obstacle in cancer treatment, mechanisms by which it develops are not well understood. Midkine (MK), a heparin-binding growth factor, has various cancer-related functions. In this study, we investigated whether MK is involved in cisplatin resistance in OSCC. We demonstrated that the Sa-3R cell line, which is OSCC cisplatin-resistant, exhibited lower MK expression with slow growth compared with its parent, Sa-3 cells. In Sa-3 cells, downregulation of MK expression significantly reduced cisplatin sensitivity, cell growth, and the expression of cyclin?D1 and cyclin?E1. MK knockdown suppressed cellular cisplatin accumulation via induction of ATP-binding cassette efflux transporters. These data suggest that MK may play important roles in cisplatin resistance in OSCC by modulating both cell growth and intracellular cisplatin accumulation.     

Authentication of newly established human esophageal squamous cell carcinoma cell line (YM-1) using short tandem repeat (STR) profiling method

Tumor Biology - Cross-contamination during or early after establishment of a new cell line could result in the worldwide spread of a misidentified cell line. Therefore, newly established cell lines...     

Schedule-dependent interactions between pemetrexed and cisplatin in human carcinoma cell lines in vitro

The combination of pemetrexed and cisplatin shows good clinical activity against mesothelioma and lung cancer. In order to study the potential cellular basis for this, and provide leads as to how to optimize the combination, we studied the schedule-dependent cytotoxic effects of pemetrexed and cisplatin against four human cancer cell lines in vitro. Tumor cells were incubated with pemetrexed and cisplatin for 24 h at various schedules. The combination effects after 5 days were analyzed by the isobologram method. Both simultaneous exposure to pemetrexed and cisplatin for 24 h and sequential exposure to cisplatin for 24 h followed by pemetrexed for 24 h produced antagonistic effects in human lung cancer A549, breast cancer MCF7, and ovarian cancer PA1 cells and additive effects in colon cancer WiDr cells. Pemetrexed for 24 h followed by cisplatin for 24 h produced synergistic effects in MCF7 cells, additive/synergistic effects in A549 and PA1 cells, and additive effects in WiDr cells. Cell cycle analysis of MCF7 and PA1 cells supported these findings. Our results suggest that the simultaneous clinical administration of pemetrexed and cisplatin may be suboptimal. The optimal schedule of pemetrexed in combination with cisplatin at the cellular level is the sequential administration of pemetrexed followed by cisplatin and this schedule is worthy of clinical investigations.