Tomoko Utsunomiya

Demethylation restores SN38 sensitivity in cells with acquired resistance to SN38 derived from human cervical squamous cancer cells

Using seven monoclonal SN38-resistant subclones established from ME180 human cervical squamous cell carcinoma cells, we examined the demethylation effects of 5-aza-2′-deoxycytidine (5-aza-CdR) on the SN38-sensitivity of the cells as well as the expression of death-associated protein kinase (DAPK) in the SN38-resistant cells. The DAPK expression levels were evaluated among parent ME180 cells, SN38-resistant ME180 cells and cisplatin-resistant ME180 cells by methylation-specific DAPK-PCR, quantitative RT-PCR and western blot analysis. The SN38-resistant cells co-treated with SN38 and 5-aza-CdR strongly exhibited enhanced SN38-sensitivities resembling those found in the parent cells. In the SN38-resistant subclones, no relationships were found between the restored SN38 sensitivity and hypermethylation of the DAPK promoter, DAPK mRNA expression, DAPK protein expression and induction of DAPK protein after 5-aza-CdR treatment, unlike the strong suppression of 5-aza-CdR-induced DAPK protein expression in the cisplatin-resistant subclones. These findings indicate that reversibly methylated molecules, but not DAPK, may regulate SN38 resistance, and that demethylating agents can be strong sensitizing anticancer chemotherapeutic drugs for SN38-resistant cancers.

3,4-Dihydroxybenzaldehyde Derived from Prunus mume Seed Inhibits Oxidative Stress and Enhances Estradiol Secretion in Human Ovarian Granulosa Tumor Cells

Granulosa cells form ovarian follicles and play important roles in the growth and maturation of oocytes. The protection of granulosa cells from cellular injury caused by oxidative stress is an effective therapy for female infertility. We here investigated an effective bioactive compound derived from Prunus mume seed extract that protects granulosa cells from hydrogen peroxide (H2O2)-induced apoptosis. We detected the bioactive compound, 3,4-dihydroxybenzaldehyde (3,4-DHBA), via bioactivity-guided isolation and found that it inhibited the H2O2-induced apoptosis of granulosa cells. We also showed that 3,4-DHBA promoted estradiol secretion in granulosa cells and enhanced the mRNA expression levels of steroidogenic factor 1, a promoter of key steroidogenic enzymes. These results suggest that P. mume seed extract may have clinical potential for the prevention and treatment of female infertility.

Impaired death-associated protein kinase-mediated survival signals in 5-fluorouracil-resistant human endometrial adenocarcinoma cells

A recent study showed that both 5-fluorouracil (5FU)-stimulated apoptosis and Fas-mediated apoptosis in human endometrial adenocarcinoma cells are enhanced by targeted knockdown of endogenous death-associated protein kinase (DAPK) with DAPK small-interfering RNAs. Therefore, we investigated the DAPK survival signals in three 5FU-resistant subclones. DAPK knockdown did not enhance 5FU-stimulated or Fas-mediated apoptosis in any of the three 5FU-resistant subclones, but the subclones acquired resistance to VP16-stimulated cell death that was DAPK-independent. Semi-quantitative flow cytometric analyses showed that there was no differential expression in nine cell surface antigens, including Fas, and six intracellular molecules, including DAPK, that may regulate cell death or survival between the parent cells and 5FU-resistant cells. DAPK mRNA and protein were expressed in the 5FU-resistant subclones at similar levels to the parent cells. These results indicate that acquisition of 5FU-resistance may be accompanied by impairment of common apoptotic signals regulating both DAPK-dependent and DAPK-independent pathways.

Direct effects of CPT-11 and SN38 on ovarian granulosa cells.

This study aimed to clarify the mechanism by which apoptosis and Fas ligand (FasL) expression are induced in the ovarian granulosa cells of mice injected with irinotecan HCl (CPT-11). To this end, the direct effects of CPT-11 and its active metabolite, SN38, on granulosa cells were investigated. Normal ovarian tissue fragments obtained from 8-week-old female MCH mice were cultured in vitro with CPT-11 or SN38 and paraffin-embedded. After sectioning, the ovarian fragments were analyzed by TUNEL staining to detect apoptotic cells and by immunohistochemistry with an anti-FasL antibody to detect FasL expression. The results revealed no increase in TUNEL-positive granulosa cells in the ovarian tissue fragments cultured with CPT-11 or SN38. Furthermore, CPT-11 and SN38 did not induce FasL expression in the ovarian fragments. In conclusion, apoptosis and FasL expression induced in the ovarian granulosa cells of mice injected with CPT-11 is not caused by direct stimulation with CPT-11 or SN38. Therefore, systemic CPT-11 administration appears to induce apoptosis and FasL expression in granulosa cells via currently unknown endogenous FasL-inducing factors or by active metabolites of CPT-11 other than SN38.