Junya Yamazaki

Overexpression of bax sensitizes human breast cancer MCF‐7 cells to radiation‐induced apoptosis

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl‐2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF‐7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation‐induced apoptosis. bax overexpression in MCF‐7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF‐7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAFI about 5‐ to 10‐fold and downregulated bcl‐2 and bcl‐XL by 80–90% at 6 hr in both parent and bax stably transfected MCF‐7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF‐7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Overexpression of Bax Sensitizes Breast Cancer MCF-7 Cells to Cisplatin and Etoposide

Bax, one of thebcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weakbax gene expression, were stably transfected withpCX2neo bax, encoding humanbax; and two unique clones,MCF-7/bax-1 andMCF-7/bax-2, that expressed different levels ofbax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level ofbax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenousbax-α overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.

OVEREXPRESSION OF BAX ENHANCES THE RADIATION SENSITIVITY IN HUMAN BREAST CANCER CELLS

Bax-a, a splice variant ofbax which promotes apoptosis, is expressed in many kinds of untransformed cell lines and breast tissue, whereas only weak or no expression could be detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weakbax gene expression, were stably transfected with pCX2neobax encoding humanbax and neomycin-resistant genes, and two unique clones (MCF-7/bax-1 and MCF-7/bax-2) were thus generated which expressed different levels ofbax-α. Sensitivity to ionizing radiation (IR) was examined and each was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level ofbax, and IR was found to induce intranucleosomal DNA fragmentation in stable transfectant but not in parent cells, thus suggesting that this sensitization is due to apoptosis. We suggest that exogenousbax-α expression might therefore be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may potentially have a therapeutic application by enhancing radiation sensitivity in breast cancer cells.

Role of milky spots as selective implantation sites for malignant cells in peritoneal dissemination in mice

We investigated the significance of milky spots for malignant cells in peritoneal dissemination using three mouse carcinomatous peritonitis models. P388 leukemia and Colon 26 cancer cells were labeled with bromodeoxyuridine (BrdU) and mice were inoculated intraperitoneally. After 24 h the greater omentum and the mesenterium were removed and stained immunohistochemically with anti-BrdU antibody. The labeled cells were found to have preferentially infiltrated into the milky spots in these specimens. Next, using B16 PC melanoma cells, which can be easily distinguished from the other cells by the intrinsic black melanin, the distribution of the melanoma cells was observed macro- and microscopically following intraperitoneal inoculation. The melanoma cells were similarly found to have selectively infiltrated into the milky spots in the omentum and mesenterium after 1 day. Moreover, the melanoma cells were growing and forming distinct metastic lesions within the milky spots 1 week later.

Inhibition of colon cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA of the Ki-ras gene

Point mutations that activate the Ki-ras proto-oncogene are present in approximately 50% of human colorectal tumors and the activated Ki-ras gene is considered to play an important role in colorectal cancer cell proliferation. Five different colon cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of Ki-ras. Treatment with antisense oligonucleotides at concentrations between 10 and 40 microM significantly and dose-dependently inhibited cell growth, colony formation and Ki-ras protein production of the colon cancer cells with activated Ki-ras, but did not affect the normal cells and colon cancer cells without Ki-ras mutation. These results show that use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behavior of human colon cancer cells with an activated Ki-ras gene.

Therapeutic effects of the angiogenesis inhibitor TNP-470 against carcinomatous peritonitis in mice.

The therapeutic effects of the new anti-angiogenesis factor TNP-470 were examined against carcinomatous peritonitis in mice. In the first experiment using carcinomatous peritonitis caused by i.p. inoculation of 10(6) M5076 tumor cells, TNP-470 solution was injected i.p. in a bolus of 50 mg/kg body weight into two groups of 10 mice either 1 or 8 days after the i.p. inoculation. The administration of TNP-470 on day 1 extended the survival time of the mice compared with 10 control mice receiving no treatment, whereas TNP-470 given on day 8 did not affect the survival time. In the next experiment on the M5076 tumor, TNP-470 solutions at 100 or 300 mg/kg were injected i.p. in a bolus into two groups of 20 mice 1 day after the inoculation 10(6) tumor cells, respectively. The administration of TNP-470 at 100 mg/kg also had an inhibitory effect. However, TNP-470 at 300 mg/kg caused toxic death in half of the mice. Next, we examined the effects of TNP-470 on another type of carcinomatous peritonitis model, which was caused by i.p. inoculation of 10(6) B16 melanoma cells. In this experiment, TNP-470 solutions in a bolus of 150 mg/kg were injected i.p. into six groups of 10 mice each on day 1 only (group 1), on days 1 and 4 (group 2), on days 1, 4 and 7 (group 3), on day 8 only (group 4), on days 8 and 11 (group 5), or on days 8, 11 and 14 (group 6), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased Apoptosis Rate by Hyperthermochemoradiotherapy for Advanced Rectal Cancers

Apoptosis induced in cancer cells by ionizing radiation, hyperthermia, and 5-fluorouracil (5-FU), termed “hyperthemochemoradiotherapy” (HCR), has been well studied in vitro; however, the role of apoptosis in the tumocidal effect of HCR for primary rectal cancers has not yet been clarified. Therefore, we examined the relationship between the therapeutic effect and induction rate of histological apoptosis in 16 patients with rectal cancers after HCR. Numerous Tunel-positive apoptotic cells were found in the tumor tissue after HCR, but few were found in the tumors which had not received HCR. The histological therapeutic effect was closely correlated to the rate of apoptosis. Thus, we suggest that HCR induces a therapeutic effect mainly through apoptosis in human rectal cancers.

CISPLATIN MICROCRYSTALS SUSPENDED IN OIL : PATHOLOGICAL STUDY OF ACUTE TOXICITY IN MICE

The pathological changes brought about by the i.p. administration of a new dosage format, clsplatin micro-crystals suspended in oil (CDDP-Oil), was examined in mice. CDDP-Oil decreased the absolute weight and the relative weight (organ weight/body weight) of the kidney, liver and spleen in the mice receiving the dosage form. However, the severity of the reduction of the organ weight in the CDDP-Oil administration groups was not different from that in the cisplatin aqueous solution (CDDP-Sol) administration groups. Histologically, severe degeneration and atrophy were recognized in the kidney, large intestine, small intestine, thymus, spleen, bone marrow and lymph nodes in the CDDP-Oil administration groups as well as CDDP-Sol administration groups. However, there were no additional changes in the macroscopic and microscopic findings in the CDDP-Oil groups. From these results, we concluded that this dosage form did not change the toxicity of cisplatin in terms of pathological effects.

NEW FORMULATION OF 5-FLUOROURACIL IN MICROSPHERES REDUCES TOXICITY IN MICE

A new dosage formulation of 5-fluorouracil incorporated in microspheres (5-FU-MS) was developed for the treatment of peritoneal carcinomatosis. We studied the acute toxicity and side effects of i.p. 5-FU-MS in mice. The 50% lethal dose value for 5-FU-MS was 535.4 mg/kg of 5-FU, which was 2.22 times that of the aqueous 5-FU solution. Deaths occurred 12–17 days after the administration of 5-FU-MS, but within 11 days after the administration of aqueous 5-FU. Thus, lethal toxicity appeared later with 5-FU-MS than with aqueous 5-FU. There were no differences in pathologic findings on autopsy between mice given the two dosage formulations.