Chizuru Sugimoto

Expression of p27 is associated with Bax expression and spontaneous apoptosis in oral and oropharyngeal carcinoma

p27Kip1, a cyclin‐dependent kinase inhibitor, is a negative regulator of the cell cycle, and apoptosis is a genetically encoded program of cell death. To clarify the relationship between the cell cycle and apoptosis, we investigated expression of p27, cyclin D1 and apoptosis‐related proteins (p53, Bax, Bcl‐2 and c‐Myc) in 60 cases of oral and oropharyngeal squamous‐cell carcinoma (SCC) using an immuno‐histochemical approach, and evaluated spontaneous apoptosis in vivo. Our most notable finding was that spontaneous apoptosis in the p27‐positive group was significantly higher than that in the p27‐negative group (p = 0.028). In addition, the percentage of p27‐positive cells was clearly correlated with that of Bax‐positive cells (γ = 0.288, p = 0.028) and with that of cyclin D1‐positive cells (γ = 0.416, p = 0.002). Expression of p27 was inversely associated with the clinical stage of total tumor progression (p = 0.027). However, no correlation was found between p27 expression and the following parameters: gender, tumor size, lymph node metastasis, overall survival and disease‐free survival. Our results give evidence that the action of the cell‐cycle regulator p27 is closely linked with apoptosis in clinical samples from patients and indicate that over‐expression of p27 might induce apoptosis in cancer cells through elevation of Bax expression, thereby acting on tumor progression. Int. J. Cancer (Pred. Oncol.) 84:315–320, 1999.

Disruption of mdr1a p-glycoprotein gene results in dysfunction of blood-inner ear barrier in mice.

P-glycoprotein (p-gp), a drug transporter in multidrug-resistant cancer cells, is a transmembrane protein encoded by mdr1a, mdr1b and mdr2 genes in mice. In our previous report, high level p-gp was immunohistochemically detected in capillary endothelial cells of the guinea pig inner ear, supporting a possible role as an extrusion pump in the blood-inner ear barrier (BIB). We investigated the functional involvement of p-gp in the inner ear using mdr1a gene knock-out mice [mdr1a(-/-) mice]. Pharmacokinetic analyses showed that mdr1a(-/-) mice displayed obviously increased accumulations of the p-gp-transported drugs doxorubicin (adriamycin, ADM) and vinblastine in the inner ear tissues compared with those in mdr1a(+/+) mice. Subsequent functional studies using auditory-evoked brainstem responses showed hearing impairment only in mdr1a(-/-) mice after administering these drugs. Furthermore, inhibition of p-gp function by co-administration of cyclosporin A (CsA) with doxorubicin (ADM) in mdr1a(+/+) mice resulted in increased accumulation of ADM in inner ear tissues and hearing impairment similar to that noted in mdr1a(-/-) mice. We conclude that mdr1a p-gp, which acts as an efflux pump in the inner ear, prevents ototoxicity induced by p-gp substrate drugs and contributes to a new functional mechanism in the BIB.

Decreased expression of Bax is correlated with poor prognosis in oral and oropharyngeal carcinoma

We investigated the expression of apoptosis-related factors, p53, Bax, Bcl-2, and spontaneous apoptosis in 57 cases of oral and oropharyngeal squamous cell carcinoma (SCC) by immunochemical staining and ApopTag kit. Positive expression of Bax was inversely associated with advanced tumor stage (P = 0.0225), lymph node metastasis (P = 0.0225), clinical stage (P = 0.0083) and poor prognosis (P = 0.0478). Positive expression of p53 was related to poor prognosis (P = 0.0445) and was associated with negative expression of Bax (P = 0.0439). The apoptosis index did not correlate with clinical outcome. These results suggest that abnormality of Bax expression plays an important role in tumor progression in oral and oropharyngeal SCC.

Conventional tumor markers are prognostic indicators in patients with head and neck squamous cell carcinoma

We tested for squamous cell carcinoma-related antigen (SCC), carcinoembryonic antigen (CEA), ferritin, immunosuppressive acid protein (IAP) and sialic acid in the serum from 247 patients with head and neck squamous cell carcinoma prior to therapy. Significant correlations were found between IAP and tumor size, lymph node metastasis, and clinical stage (P<0.0001, P<0.001, and P<0.0001). Also, sialic acid and SCC were also correlated with tumor size, lymph node metastasis, and clinical stage. Moreover IAP, sialic acid and SCC were strongly associated with survival rate (P<0.0001, P = 0.0230 and P = 0.0159). A multivariate Cox proportional hazard model demonstrated that being positive for IAP was an independent predictor for patients with H&NSCC (P = 0.0115). The results indicate that IAP, sialic acid and SCC are useful as prognostic factors.

Apoptosis‐promoting gene (bax) transfer potentiates sensitivity of squamous cell carcinoma to cisplatin In vitro and In vivo

Modulation of apoptosis may potentiate the sensitivity of tumor cells to chemotherapeutic agents, thus improving the clinical outcome of cancer treatment. Bax, an apoptosis‐promoting member of the bcl‐2 family, may be a key factor influencing the chemosensitivity of tumor cells, however, its involvement in cellular sensitivity to anti‐cancer drugs remains uncertain in squamous cell carcinoma (SCC). To investigate the role of bax gene expression in modulating cisplatin (CDDP)‐induced apoptosis in vitro, an established CDDP‐resistant human head and neck SCC (IMC‐3 cell line) was transfected with bax gene‐bearing mammalian expression vector. Overexpression of the bax gene in CDDP‐resistant IMC‐3 cells elevated the CDDP susceptibility of tumor cells to a level similar to that of the parental IMC‐3 cells. In an in vivo study, percutaneous transfer of apoptosis‐promoting bax gene by particle‐mediated (gene gun) delivery caused overexpression of Bax in SCC, which was confirmed by immunohistochemical staining, and inhibited the growth of mouse CDDP‐resistant SCC. Furthermore, combination therapy with bax gene transfer and subcutaneous administration of CDDP at 3‐day intervals markedly inhibited the growth of mouse SCC. Thus, overexpression of bax in SCC by a gene gun system appears to be a rational approach to improving the efficacy of chemotherapy and treatment outcome. We suggest that exogenous bax expression may have therapeutic applications for enhancing chemotherapy in SCC. Int. J. Cancer 82:860–867, 1999.

Granulocyte colony-stimulating factor (G-CSF)-mediated signaling regulates type IV collagenase activity in head and neck cancer cells.

Granulocyte colony‐stimulating factor (G‐CSF), a hematopoietic cytokine, regulates the proliferation and differentiation of granulocytic progenitor cells and functionally activated mature neutrophils. G‐CSF also affects nonhematopoietic tumor cells through its binding to the specific receptor (G‐CSFR) on the cells. The type IV collagenase [matrix metalloproteinase 2 (MMP‐2)] is known to play a main role in the process of invasion and metastasis, but its regulation, for example, in expression or in activation, is not clearly understood. In this study, we investigated the role of G‐CSF in the regulation of tumor cell invasion and the synthesis of MMP‐2. G‐CSFs producing the head and neck carcinoma cell line T3M‐1 cells with metastatic ability and no G‐CSF receptor (G‐CSFR) expression were transfected with G‐CSFR expression vector. In vitro treatment of G‐CSFR‐transfectant T3M‐1 cells with recombinant G‐CSF (rG‐CSF) significantly augmented their invasive potential in a reconstituted basement membrane (Matrigel) system compared with that of parental cells. Moreover, MMP‐2 activity of G‐CSFR‐transfectant T3M‐1 cells was enhanced by the stimulation with rG‐CSF, as assessed by gelatin zymography. These results identify G‐CSF as a regulator of MMP‐2 and cellular invasion.

Timing for removal of tympanic ventilation tube in children

The medical records of 220 ears of 137 pediatric patients (85 male and 52 female) in which three kinds of ventilation tubes were inserted for treating otitis media with effusion (OME) were reviewed. The tubes selected were the Shepard grommet (75 ears), Goode-T (39 ears), and Paparella type II tube (106 ears). The criteria for tube placement were as follows: (1) continuous conductive hearing loss with over 25 dB air-bone gap, (2) resistance to conservative therapy for over 6 months, and (3) retracted and glue-colored tympanic membrane with type B tympanogram. The tubes that remained in place for over 18-24 months were removed intentionally in combination with a freshening of the perforation edge and tape-patch technique using Steri-Strip tape (3M) for preventing permanent eardrum perforation, because the incidence of persistent perforation became higher after long-term intubation. Shepard grommets tended to be extruded earlier, while Paparella type II tubes tended to stay longer. The OME recurrence rate decreased 12 months or more after tubal insertion. There was a tendency for the recurrence rate to decrease the longer the tube stayed in the eardrum. The number of recurrences decreased when the patients age at the tube removal or extrusion was 7-8 years old. Adenoidectomy did not influence the recurrence rate of OME. Although the Goode-T and Paparella tube II tubes showed high perforation rates, the perforation rate after extrusion or removal of the tube was decreased by the use of the tape patch technique in combination with a freshening of the perforation edge. From these findings, it was concluded that the appropriate intubation period for the treatment of OME in children is over 12 months with the use of a long-term tube, and that if the patients age at the time of tube insertion was below 6 years, it might be better that the removal of the tube is postponed until the patient is 8 years of age.

Expression of hMSH2 correlates with in vitro chemosensitivity to CDDP cytotoxicity in oral and oropharyngeal carcinoma

We investigated the expression of hMSH2, a human mutS homologue from chromosome 2p, in oral and oropharyngeal squamous cell carcinoma (SCC) by an immunohistochemical technique and performed tumor in vitro chemosensitivity testing. In 58 oral and oropharyngeal SCC, the hMSH2 positive score was inversely associated with tumor size, but not with other clinical parameters. Among five anticancer drugs (cisplatin (CDDP), 5-FU, peplomycin, mitomycin C and doxorubicin), only for CDDP was sensitivity to cytotoxicity correlated with the hMSH2 positive score. The susceptibility of hMSH2-positive tumors to CDDP killing was significantly higher than that of hMSH2-negative tumors. Immunohistochemical results regarding hMSH2 are promising in the evaluation of the sensitivity of cancer cells to CDDP cytotoxicity and enable one to select patients for adjuvant chemotherapy for oral and oropharyngeal SCC.

Enhancement of cisplatin sensitivity and platinum uptake by 40 °C hyperthermia in resistant cells

Abstract We studied the cytotoxic and pharmacological properties of 40 °C hyperthermia and CDDP in CDDP-sensitive (IMC-3) and CDDP-resistant (IMC-3-DDP) human maxillary carcinoma cells. Heating at 40 °C alone caused almost no cell killing to IMC-3 and IMC-3-DDP cells. In both cell lines, the dose-dependent cytotoxicity of 2-h exposures to CDDP was increased at 40 °C in comparison to 37 °C. Heating at 40 °C also potentiated CDDP cytotoxicity in both IMC-3 and IMC-3-DDP cells with thermal chemoenhancement ratios (CER) of 1.48 and 1.94, respectively. The intracellular CDDP uptake level of IMC-3-DDP at 37 °C was significantly reduced compared with IMC-3 cells. At 40 °C, however, hyperthermia increased platinum accumulation by factors of 1.4 and 1.8 in IMC-3 and IMC-3-DDP cells, respectively. These findings indicated that CDDP sensitivity was hyperthermically chemopotentiated in CDDP-resistant variants rather than in the control clones. Thus, clinical cancer chemotherapy with CDDP may be improved by an appropriate combination with hyperthermia even at 40 °C.

Expression of Fas (CD95) ligand is correlated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal squamous cell carcinoma

The Fas/Fas ligand (FasL) pathway has been shown to be an important cellular pathway mediating apoptosis. In this study we investigated the expression of Fas and FasL and the rate of spontaneous apoptosis in 58 oral and oropharyngeal squamous cell carcinoma (SCC) by using immunohistochemical techniques. There was no correlation between Fas or FasL expression and clinicopathological factors. The expression of Fas in the tumor did not affect spontaneous apoptosis of the tumor cells. However, FasL expression was associated with IL-10 and granulocyte colony-stimulating factor expression in oral and oropharyngeal SCC. These results suggested that the Fas/FasL system is connected with the expression of various factors including cytokines in tumor cells.