Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA.

Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV small-interfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPV-positive cervical cancer.

Down-regulation of the insulin-like growth factor I receptor by antisense RNA can reverse the transformed phenotype of human cervical cancer cell lines.

The insulin-like growth factor I receptor (IGF-IR) plays an essential role in the establishment and maintenance of transformed phenotype, and interference with the IGF-IR pathway by antisense or dominant-negative mutants causes reversal of the transformed phenotype in many rodent and human tumor cell lines. We stably transfected an IGF-IR antisense mRNA expression plasmid into human papillomavirus (HPV)-negative C33a cell line, HPV-16-positive SiHa cell line, and HPV-18-positive HeLa S3 cell line to determine whether the IGF-IR could be a target for cervical cancer cells, especially in the presence of HPV. Approximately 30-80% down-regulation of IGF-IR expression was observed by Western blot in antisense transfected clones. There was a little inhibition in monolayer growth in all cell lines. In C33a cells, wild-type and sense clones formed 92-146 colonies in soft agar after 3 weeks; antisense clones formed <12 colonies. In SiHa cells, wild-type and sense clones formed approximately 60 colonies after 5 weeks; antisense clones formed 0-3 colonies. In HeLa S3 cells, wild-type and sense clones formed 218-291 colonies in soft agar after 2 weeks; antisense clones formed 14-160 colonies. There was a good correlation between IGF-IR down-regulation level and inhibition of transformation in soft agar. Tumorigenesis in nude mice was strongly inhibited in HeLa S3 and SiHa clones transfected with the antisense. These results indicate that down-regulation of IGF-IR by antisense RNA can reverse the transformed phenotype of human cervical cancer cells, even when harboring malignant type HPVs.

Thrombospondin-1 and -2 messenger RNA expression in invasive cervical cancer: correlation with angiogenesis and prognosis.

PURPOSE: TSP association with clinicopathological features, including microvessel count, regarding prognostic significance was examined in patients presenting with invasive cervical cancer. EXPERIMENTAL DESIGN: Gene expression of TSP-1 and TSP-2 was assessed by reverse transcription-PCR in 10 normal cervix and 78 invasive cervical cancer samples. RESULTS: TSP-1 and TSP-2 mRNA expression was detected in seven (70.0%) of the normal cervical specimens. TSP-2 mRNA expression in normal cervix was significantly higher than that in cases involving cervical cancer (P = 0.032). TSP-1 mRNA expression was significantly lower in tumors characterized by advanced stage (P = 0.047). Fifty-three patients displaying stage Ib-IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. Expression of TSP-1 and TSP-2 mRNA was significantly lower in tumors exhibiting parametrial invasion (P = 0.016 and P = 0.049, respectively). Microvessel counts were significantly higher when decreased TSP-1 expression was evident (P = 0.029). The microvessel count in patients lacking TSP-2 mRNA expression was higher than that observed in patients displaying TSP-2 mRNA expression, although it was not statistically significant (P = 0.062). Subjects demonstrating TSP-1 mRNA expression exhibited significantly better prognosis than those lacking TSP-1 mRNA expression (P = 0.0038). Furthermore, TSP-1 mRNA expression was an independent prognostic factor in the multivariate analysis. CONCLUSIONS: These findings provide evidence that TSP-1 expression is of value as a prognostic factor in cervical cancer. The inverse correlation between TSP expression and microvessel count also indicates that decreased TSP expression may be associated with an angiogenic phenotype in this class of neoplasm.

Presence of human papilloma virus DNA in pelvic lymph nodes can predict unexpected recurrence of cervical cancer in patients with histologically negative lymph nodes.

Patients without any evidence of lymph node metastases are supposed to have a fair prognosis, but some of these patients develop recurrent disease unexpectedly after surgery. The object of this study is to examine whether the detection of human papilloma virus (HPV) DNA could be used as a diagnostic marker to predict such recurrences. Two hundred and thirty-six patients undergoing radical hysterectomy and pelvic lymphadenectomy for stage Ib and II cervical cancer at Okayama University Hospital (Japan) from 1988-1994 were reviewed, and only those cases positive for HPV-16 or HPV-18 in primary sites were included in this survey. The E6-E7 region of the HPV genome was amplified by a sensitive nested PCR from archival pelvic lymph node specimens. HPV sequences identical to those of the primary sites were detected in histologically confirmed negative lymph nodes, regardless of histological type or HPV type of the primary lesion, in 9 of 10 patients who recurred within 4 years of surgery. In contrast, histologically confirmed negative lymph nodes from 12 patients with stage IIb disease without evidence of recurrent disease were all negative for the presence of HPV, except for 1 lymph node. The presence of HPV DNA in histologically negative nodes implies the possibility of early nodal involvement or coexistence of undetectable hematogenic dissemination and could therefore be used as a diagnostic marker to predict the unexpected recurrence of these patients.

Basic fibroblast growth factor stimulates DNA synthesis in cells overexpressing the insulin-like growth factor-I receptor.

Insulin-like growth factor-I (IGF-I), by itself, cannot sustain the growth of BALB/c 3T3 cells, but requires the cooperation of other growth factors, such as platelet-derived growth factor or epidermal growth factor. In 3T3 cells constitutively overexpressing the human IGF-I receptor, called p6 cells, IGF-I by itself is fully mitogenic. We show here that p6 cells are also stimulated to enter DNA synthesis by the sole addition of basic fibroblast growth factor (bFGF), which, by itself, is incapable of stimulating parental 3T3 cells. Although bFGF does not bind directly to the IGF-I receptor, it induces its autophosphorylation. Stimulation of p6 cells by bFGF is not inhibited by an antibody to the IGF-I receptor that inhibits IGF-I-mediated DNA synthesis, and IGF-I is not detectable in the medium of bFGF-treated p6 cells. Stimulation cannot be explained by an increased number of FGF receptors, because p6 cells actually have slightly fewer FGF receptors than parental BALB/c 3T3 cells. Basic FGF also stimulates DNA synthesis in 3T3 cells overexpressing a mutant IGF-I receptor that does not autophosphorylate in response to IGF-I and has lost its mitogenic potential. Although we were unable to demonstrate directly that bFGF causes transphosphorylation of the IGF-I receptor, we conclude that in cells overexpressing the IGF-I receptor, bFGF can stimulate DNA synthesis either by an unknown mechanism or through transphosphorylation of the IGF-I receptor.

The role of the IGF-1 receptor in the stimulation of cells by short pulses of growth factors.

Balb/c 3T3 cells require prolonged stimulation by serum or growth factors to enter DNA synthesis. However, in p6 cells, a derivative cell line from 3T3 cells which constitutively over-express the insulin-like growth factor-1 (IGF-1) receptor, a serum pulse of only 1 h is sufficient for maximal stimulation. Furthermore, maximal stimulation of DNA synthesis is also obtained when 3T3 cells, serum-stimulated for only 1 h, are subsequently incubated with IGF-1. Our results indicate that short pulses of growth factors render 3T3 cells capable of responding to IGF-1, either by increasing the number of IGF-1 receptors or by providing a new substrate for the activated receptor.

Vascular endothelial growth factor and platelet-derived endothelial cell growth factor expression are implicated in the angiogenesis of endometrial cancer.

Although many angiogenic factors have been described, it is not well defined which factors are expressed in endometrial cancer. The object of this study was to examine mRNA levels of the two angiogenic factors, vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF) in endometrial cancer tissues and their association with clinicopathological features including microvessel density. The level of VEGF and PD-ECGF mRNAs was assessed by semi-quantitative reverse transcription-polymerase chain reaction using beta-actin as an internal standard in 38 patients with endometrial cancer. Microvessel counts were also assessed by immunostaining for factor VIII-related antigen in the most vascularised area of the specimen. VEGF/beta-actin ratios of non-endometrioid tumours were significantly higher than those of endometrioid tumours (P = 0.013). VEGF/beta-actin ratios of cases with lymph-vascular space involvement were significantly higher than those of cases without lymph-vascular space involvement (P = 0.021). Although it was not statistically significant, PD-ECGF/beta-actin ratios in grade 3 tumours were higher than those in grade 1 and 2 tumours (P = 0.066). The microvessel density was significantly correlated with the level of VEGF and PD-ECGF mRNA expression (P = 0.041 and P < 0.0001, respectively). Our findings provide evidence that the expression of both VEGF and PD-ECGF is involved in the promotion of angiogenesis in endometrial cancer. In addition, VEGF and PD-ECGF might contribute to the aggressive potential of high grade tumours or certain histological subtypes with unfavourable prognosis through the induction of angiogenesis.

Vascular endothelial growth factor is implicated in early invasion in cervical cancer.

The association between the expression of vascular endothelial growth factor (VEGF) and clinicopathological factors has scarcely been examined in cervical cancer. This study examines the level of VEGF messenger RNA (mRNA) expression in invasive cervical cancer and its association with clinicopathological features including microvessel density. The level of VEGF mRNA was assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta-actin as an internal control in 66 patients with stages Ia-IVb invasive cervical cancer. In 42 patients who underwent surgery, the microvessel count was also assessed by immunostaining for factor VIII-related antigen in the most neovascularised area of the specimen. The highest level of VEGF mRNA expression was observed in early invasive cervical cancers. Except for stage IVb, the stage of the disease inversely correlated with the level of VEGF mRNA (P < 0.05). There was no significant difference in the level of VEGF mRNA with respect to histological cell types. 38 patients with stages Ib-IIb cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. There was no significant difference in the level of VEGF mRNA with respect to lymph node metastasis, depth of stromal invasion, tumour size, parametrial involvement or vaginal involvement among these patients. A significant relationship was found between the microvessel density and the level of VEGF mRNA (P < 0.01). These findings provide evidence that the expression of VEGF is involved in the promotion of angiogenesis in cervical cancer and plays an important role in early invasion.

CD44 exon v6 correlates with cellular differentiation but not with progression and metastasis of cervical cancer.

The purpose of this study was to investigate whether CD44v6 expression correlates with progression or metastasis of cervical cancer. The presence of mRNA for CD44v6 was examined, the association with clinicopathological features was assessed in 80 patients with cervical cancer by reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent Southern blot hybridisation with an oligonucleotide probe specific for v6. The standard form of CD44 was expressed in all specimens and 53 of 80 cervical cancers expressed an isoform containing exon v6 in combination with other variant exons. In addition, longer size transcripts of more than 1350 bp (long form) were identified in 22 of the 53 CD44v6 positive patients. The expression of CD44v6 and CD44v6 long form in squamous cell carcinomas was significantly higher than that in non-squamous cell carcinomas (P < 0.001). The expression of CD44v6 long form in histological grade 1 and 2 was significantly higher than that in grade 3 (P < 0.05). 47 patients in stage Ib-IIb cervical cancers were treated by radical hysterectomy and pelvic lymphadenectomy. We did not find any association between the expression of the long form or the short form of CD44v6 and any pathological features, except for histological cell type. These findings suggest that the regulation of CD44v6 seems to be different between different histological cell types and different tumour grades, and the expression of CD44v6 might not be implicated in the progression and metastasis of cervical cancer.

CD44 exon v6 is not implicated in the progression and metastasis of endometrial cancer.

We examined the presence of mRNA for CD44v6 and assessed the association with clinicopathological features in 42 patients with endometrial cancer by RT-PCR and subsequent Southern blot hybridization with oligonucleotide probe specific for v6. The standard form of CD44 was expressed in all specimens and 20 out of 42 endometrial cancers expressed an isoform containing exon v6 in combination with other variant exons. However, there was no correlation between the expression of CD44v6 and any clinicopathological factors. These findings suggested that the expression of CD44v6 is not implicated in the progression and metastasis of endometrial cancer.

Alteration of the CDKN2/p16 gene is not required for HPV-positive uterine cervical cancer cell lines.

To determine whether alterations of the CDKN2/p16 might be involved in HPV-positive cervical cancers, we examined for alterations of this gene and function of the protein p16 to interact with CDK4 in 5 cervical cancer cell lines. No alteration of this gene was detected. Proteins for p16 and CDK4 were normally expressed and function of p16 to interact with CDK4 was not abrogated in these cell lines. These cell lines were human papillomavirus (HPV)-positive and carried wild-type p53. These findings suggest that phosphorylation of pRb by CDK4 is not critical in the carcinogenesis or in the establishment of HPV-positive cervical cancer cell lines, since HPV E6 or E7 viral-transforming proteins inactivate p53 and pRb tumor suppressor protein function, resulting in deregulated progression of the cell cycle.

Detection of c-erbB-2 and FGF-3 (INT-2) gene amplification in epithelial ovarian cancer.

We examined gene amplifications of the c-erbB-2 and FGF-3 gene in 48 epithelial ovarian cancers by differential PCR and addressed their association with clinico-pathological features including clinical outcome. Overall, 25.0 and 20.8% of ovarian cancers displayed amplified c-erbB-2 or FGF-3 gene, respectively. Amplification of the c-erbB-2 gene was significantly associated with particular histological cell types, higher histological grade, and low levels of serous CA125. However, there was no correlation between c-erbB-2 gene amplification and patient outcome. No correlation was observed between FGF-3 gene amplification and any clinico-pathological features including overall survival. These findings suggested that c-erbB-2 or FGF-3 gene amplification might be one of the oncogenic events implicated in the development of ovarian cancers, yet is not a useful prognostic marker.

Angiogenesis and platelet-derived endothelial cell growth factor/thymidine phosphorylase expression in endometrial cancer.

The object of this study was to clarify the association of angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (dThdPase) with clinicopathological factors including tumor angiogenesis and patient outcome in endometrial cancer. There was no correlation between the expression of PD-ECGF in cancer cells and any of the clinicopathological variables. Immunopositivity for PD-ECGF in stroma cells was significantly higher in poorly differentiated adenocarcinomas. The microvessel counts correlated with PD-ECGF positive stroma cells (p<0.0001). Disease-free survival was significantly worse in patients with marked PD-ECGF expression in stromal cells and high microvessel count. A multivariate analysis using Cox's proportional hazard model showed that high microvessel counts independently predicted disease-free survival as well as stage and myometrial invasion. The expression of PD-ECGF in stroma cells may play a crucial role in the promotion of angiogenesis. Tumor angiogenesis can be used to predict prognosis in patients with endometrial cancer.

Angiogenesis and platelet-derived endothelial cell growth factor/thymidine phosphorylase expression in cervical cancer.

The object of this study was to clarify the association of platelet-derived endothelial cell growth factor (PD-ECGF)/thymidine phosphorylase (dThdPase), separately assessed in cancer cells and in stroma cells, with clinicopathological factors including tumor angiogenesis and prognosis in cervical cancer. The expression of PD-ECGF was evaluated by immunohistochemical staining in 92 patients with stage Ib-II cervical cancer. The microvessel count was assessed by immunostaining for factor VIII-related antigen in the most neovascularized area. Microvessel count was significantly higher in tumors with non-squamous cell carcinoma. PD-ECGF expression in cancer cells was significantly higher in tumors with pelvic node metastasis and squamous cell carcinoma. Immunopositivity for PD-ECGF in stroma cells was significantly higher in tumors with large size and deep stromal invasion. The microvessel counts in cases with positive PD-ECGF expression in stroma cells were significantly higher than those in cases with negative PD-ECGF expression in stroma cells (p=0.048). Disease-free survival and overall survival were significantly worse in patients with deep stromal invasion, parametrial involvement, vaginal involvement, lymph-vascular space involvement, pelvic lymph node metastasis and high microvessel count. A multivariate analysis using Cox's proportional hazard model showed that high microvessel count independently predicted disease-free and overall survival. The expression of PD-ECGF in stroma cells may play a crucial role in the promotion of angiogenesis and tumor angiogenesis can be used as a useful prognostic marker for cervical cancer.

Thrombospondin-1 and -2 messenger RNA expression in normal and neoplastic endometrial tissues: correlation with angiogenesis and prognosis.

The role of thrombospondin (TSP) in tumor angiogenesis and progression remains controversial. The expression of TSP-1 and TSP-2 mRNAs was assessed. Furthermore, TSP association with clinicopathological features, including microvessel count, regarding prognostic significance was examined. Expression of TSP-1 and TSP-2 were assessed by reverse transcriptase-polymerase chain reaction in 18 normal endometrium and 55 endometrial cancer samples. Microvessel counts were determined by immunostaining for factor VIII-related antigen in endometrial cancer specimens. TSP-1 expression of secretory phase endometrium was markedly higher than that of proliferative phase endometrium (p=0.047). Expression of TSP-1 and TSP-2 was detected in 33 (60.0%) and 15 cases (27.3%), respectively, of 55 endometrial cancer samples. TSP-1 expression was significantly higher in tumors recovered from elderly women (p=0.009). TSP-2 expression was significantly higher in malignancies exhibiting cervical and lymph-vascular space involvement (p=0.029 and p=0.009, respectively). Although not statistically significant, microvessel counts were higher in cases displaying increased TSP-1 expression. The microvessel count in patients with TSP-2 expression was markedly higher than that observed in patients lacking TSP-2 expression (p=0.026). Subjects demonstrating TSP-2 mRNA expression displayed significantly poorer prognosis than those lacking TSP-2 mRNA expression (p=0.016). There was no association between TSP-1 mRNA expression and patient outcome. Our findings provide evidence that elevated TSP expression may be associated with an angiogenic phenotype in endometrial cancer. In addition, TSP-2 expression is a marker for poor prognosis in this disease.

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Cytocidal effect and DNA damage of nedaplatin in vitro by simulating pharmacokinetic parameters.

PURPOSE: The pharmacodynamic effects of cis-diammine(glycolato)platinum (nedaplatin, 254-S) in vitro have been reported, but the dosage and exposure time in vitro have not always been based on clinical observations of the drug's actions in vivo. Regardless of the actual exposure conditions used, the effect of cell-cycle nonspecific anticancer agents such as nedaplatin is believed to depend on the area under the drug concentration-time curve (AUC). In this study, we evaluated the pharmacodynamics of nedaplatin in vitro, especially in relation to its AUC dependency, in terms of cell survival and DNA crosslinking. METHODS: BG-1 human ovarian cancer cells were treated with various concentrations of nedaplatin to simulate the pharmacokinetics of administration in a clinical setting. The BG-1 cells were exposed to nedaplatin dissolved in medium containing serum using constant concentration conditions, either high (maximum 7.69 mg/l) or low (average 1.33 mg/l). These concentrations were based on doses used in clinical studies. We then adjusted the exposure conditions in vitro to simulate the elimination of the drug from serum in vivo as follows: T1/2 alpha 1.20 h and T1/2 beta 2.70 h. The AUC values were set at 4, 8, 16, 25 and 40 mg.h/l for all exposure conditions. A colony-formation assay for the surviving fraction and an alkaline-elution assay for DNA crosslink measurement were done for the pharmacodynamic evaluation with comparison on the basis of the AUC value. RESULTS: Exposure to a low concentration for a long time was the most effective of the exposure conditions at the same AUC value. The greater the AUC value, the higher the crosslink index under all exposure conditions. This index tended to increase particularly after exposure to the low concentration. The natural logarithm of the surviving fraction (Y') was a linear function of the crosslink index regardless of the drug-exposure condition: ln(Y') = -87.2x + ln(5.79), R2 = 0.89. The threshold cytocidal effect was associated with a crosslink index of 0.02. CONCLUSION: There was a strong correlation between the cytocidal effect of nedaplatin and DNA crosslink formation. The cytocidal effect and DNA crosslinking in vitro depended on the exposure conditions used to define the AUC. Therefore, a new pharmacokinetic-pharmacodynamic model for nedaplatin must be constructed to investigate the most effective administration procedure in vivo.

Cytocidal effect and DNA damage of nedaplatin: a mathematical model and analysis of experimental data.

PURPOSE: Cell cycle non-specific anticancer agents such as cis-diamminedichloroplatinum(II) are believed to depend linearly on the value of the area under the drug concentration time curve, which is supported by a mathematical model. However, the quantitative non-linear phenomena of both the cytocidal effect and DNA crosslink formation by cisdiammine(glycolato)platinum (nedaplatin) have been shown in vitro. Therefore, we developed a new mathematical model to explain these phenomena. METHODS: We assumed that nedaplatin enters intracellular fluid from medium through simple diffusion to form DNA crosslinks that kill cells. We developed a mathematical model to represent this assumption using differential equations that we then solved using an original computer program. The calculated results were compared with the experimental data. RESULTS: The drug's simple diffusion rate constant, the DNA crosslink formation rate constant, and the crosslink-dependent cell death rate constant in the model were 1.8 x 10(-14) (l h-1), 1.6 x 10(8) (l mol-1/2 h-1), 5.45 x 10(1) (mol-1), respectively. The model fits the experimental results statistically. The model also demonstrated theoretical proof that continuous exposure at a low dose was superior to the short exposure at a high dose seen in published experimental data. CONCLUSIONS: We developed a mathematical model to describe the non-linear pharmacodynamic effect of nedaplatin in vitro. This model may provide a novel drug infusion procedure for cancer patients.

Platelet-derived endothelial cell growth factor is not implicated in progression of cervical cancer.

We examined the expression of platelet-derived endothelial cell growth factor (PD-ECGF) mRNAs in 47 invasive cervical cancer tissues by semi-quantitative RT-PCR and addressed its association with clinicopathological features including microvessel density. Squamous cell carcinomas expressed higher levels of PD-ECGF mRNA than non-squamous cell carcinomas (P=0.014). We found no association between FIGO stage and levels of PD-ECGF mRNA. A subset of 31 patients with stage Ib-II cervical cancer underwent radical hysterectomy and pelvic lymphadenectomy. There were no significant differences in PD-ECGF mRNA levels with respect to tumor size, degree of stromal invasion, lymphvascular space involvement, parametrial involvement, vaginal involvement or lymph node metastasis among these patients. There was no correlation between microvessel density and the levels of PD-ECGF mRNA. These findings suggest that PD-ECGF expression is not associated with progression and metastasis of cervical cancer.

Macrophage infiltration and angiogenesis in endometrial cancer.

The aim of this study was to determine whether tumor-associated macrophages (TAMs) infiltration correlates with clinicopathological factors including microvessel, counts and clinical outcome in endometrial cancer. Overall 56 out of the 109 endometrial cancers (51.4%) expressed distinct tumor-associated macrophages infiltration in their tumor stroma. Tumor-associated macrophages infiltration was significantly high in tumors with deep myometrial invasion, high grade and elderly patients. Microvessel counts strongly correlated with tumor-associated macrophages infiltration in tumor stroma (p = 0.0002). However, tumor-associated macrophages infiltration was not a prognostic factor. In conclusion, tumor-associated macrophages may play a crucial role in the promotion of angiogenesis, but can not be used to prodict prognosis of patients with endometrial cancer.