Jang-In Shin

Photodynamic therapy with 9-hydroxypheophorbide α on AMC-HN-3 human head and neck cancer cells: Induction of apoptosis via photoactivation of mitochondria and endoplasmic reticulum

Skin phototoxicity is the main side effect of photodynamic therapy (PDT). To overcome this problem, some new photosensitizers have been developed with longer absorbance wavelengths and shorter half-life in the body. In this study, we investigated the mechanism of PDT mediated by a new chlorophyll derivative photosensitizer, 9-hydroxypheophorbide α (9-HPbD), on AMC-HN-3 cancer cells. Phototoxicity and apoptosis on AMC-HN-3 cells induced by 9-HPbD was exhibited in a time- and dose-dependent manner. Mitochondria and endoplasmic reticulum (ER) were observed as preferential sites of 9-HPbD accumulation. Photoactivation of 9-HPbD-loaded AMC-HN-3 cells led to a rapid generation of reactive oxygen species (ROS) at 30 min, followed by a loss of mitochondrial membrane potential (MMP) at 2 h, translocation of apoptosis-inducing factor (AIF) at 2 h, and the release of cytochrome c at 3 h following PDT. Caspase-12, an important caspase involved in ER-induced apoptosis, and C/EBP homologous protein (CHOP), an ER stress inducible transcription factor, were also up-regulated after PDT (3-12 h and 6-12 h, respectively). Subsequently, activation of caspase-9 at 6 h, caspase-3 and PARP at 12 h also occurred in PDT-treated AMC-HN-3 cells. The above observations demonstrate that both mitochondria and ER serve not only as the sites of sensitizer binding, but also the subcellular targets of 9-HPbD-PDT, effective activation of which is responsible for 9-HPbD PDT-induced apoptosis in AMC-HN-3 cells.

Possible link between no concentrations and COX-2 expression in systems treated with soy-isoflavones

Abstract:  The production of nitric oxide (NO) emerges as an essential determinant in auto‐ and paracrine signaling. NO is known to be generated under inflammatory conditions, carcinogenesis, and circulatory shock. The large amount of NO produced in response to cytokines plays an important role in inflammatory conditions. Cyclooxygenase (COX), the central enzyme in prostanoid biosynthesis, is involved in the first step of prostanoid synthesis from arachidonic acid. The reported studies to evaluate the relationship between NO and COX‐2 have revealed both inhibitory and stimulatory effects of NO on COX‐2 expression. Genistein, one of soy‐isoflavones, is a polyphenolic flavonoid and a potent antioxidant and anti‐inflammatory agent. In the present article, the effect of soy‐isoflavones on NO production and COX‐2 gene expression was examined. NO production by soy‐isoflavones was greatly increased even though eNOS and iNOS expression were not different from nontreated control. The increment of NO was accompanied with the elevated expression of COX‐2 and the concentrations of PGE2. The COX‐2 stimulatory effect of soy‐isoflavones appeared to be modulated by ERK‐1 and ‐2 and p38. In mammalian cancer system, incubation with the NO donor sodium nitroprusside (SNP) resulted in a slight upregulation of COX‐2, and cotreatment with genistein decreased COX‐2 expression possibly by the activation of AMP‐activated protein kinase (AMPK).

An in vitro and in vivo study of combination therapy with Photogem®-mediated photodynamic therapy and cisplatin on mouse cancer cells (CT-26).

OBJECTIVE This study was designed to evaluate the anticancer effect of cisplatin and photodynamic therapy (PDT) combined in vitro and in vivo. BACKGROUND DATA PDT, these days, is a promising modality for the treatment of cancer and infections. In order to optimize the treatment, cisplatin is often combined with other chemotherapeutic agents. METHODS Colon cancer cells were incubated with cisplatin (0.1, 1, and 6 μg/ml), followed by photosensitization with Photogem® and irradiation with a 632 nm diode laser at an energy density of 3.2 J/cm(2). An MTT assay was then used to measure cell viability. For in vivo studies, established tumors were treated with cisplatin (3 mg/kg) alone or with PDT (5 mg/kg of Photogem®, 600 J/cm(2)). The sizes of the tumors were continuously measured to note the effects. RESULTS The cell viability of the combined therapy group was 19.88 ± 0.41, corresponding to a 9% increase compared with that of the cisplatin- or PDT-only groups. In vivo, the tumors treated with PDT or combination therapy disappeared completely three days after each treatment, but on the 14th day, the recurrence rate was significantly lower in the combination therapy group than in the PDT group. CONCLUSIONS Combination therapy results in an enhanced anticancer effect, presenting the possibility of minimizing the administration dosage of Photogem® and cisplatin.

Proapoptotic potentials of genistein under growth stimulation by estrogen.

Abstract: In mammary carcinogenesis, hormonal effects have been reported to be important factors. Estrogens are known to regulate the proliferation of breast cancer cells, whereas genistein has been shown to induce apoptosis in mammary tumor cells. This study examined genistein‐induced apoptosis through the regulation of bcl‐2 and bax expression in the presence of estrogen. MCF‐7 cells were treated with either genistein (25, 50, and 100 μM) or in the presence of 17β‐estradiol (12.5, 25, and 50 nM) for 48 h. DNA ladder analysis and Western blot analysis of bcl‐2, bax, cyclin B1, p21, and p53 were carried out. For comparison, the in vivo system was employed using estrogen‐deficient and estrogen‐sufficient female rats at two different concentrations of genistein. In MCF‐7 cells, DNA fragmentation was evident by the treatment of genistein in the absence and presence of estrogen. Downregulation of bcl‐2 and upregulation of bax by genistein were observed. However, genistein showed no proapoptotic properties in the presence of estrogen except with the lowest concentration of estrogen. In the presence of estrogen, p21 and p53 protein expression were upregulated by high concentrations of genistein. Bcl‐2/bax ratios were decreased by genistein treatment in the presence or absence of estrogen in female rats. These results demonstrate that the proapoptotic property of genistein might be influenced greatly by the concentration of estrogen in vitro, but that this influence by estrogen is not evident in vivo.

Hydroxypheophorbide-α-mediated photodynamic therapy augmented by pretreatment with genistein in CaSki cervical cancer cells

Photodynamic therapy (PDT) is a treatment for cancer involving three key components — a sensitizing compound (light) tissue, and oxygen. In this study we applied phototreatment to cancer cells with 2 J.cm-2 of red light after sensitizing with 9-hydroxypheophorbide-α (9-HpbD-α), a new chlorophyll-derived photosensitizer. We have investigated the cytotoxic and apoptotic effects of 9-HpbD-α-induced PDT in cervical cancer cells, the enhancing effect of genistein in PDT, and explored the molecular mechanisms of E6 or E7 oncogenes, apoptotic signaling molecules, and ER stress. Co-treatment downregulated the transcripts of the E6*I, E6*II, and E7 oncogenes. Combined treatment with PDT and genistein showed typical apoptotic features, i.e. apoptotic bodies. To elucidate the mechanism of combination treatment-induced apoptosis, various mediators of apoptosis were investigated. Activation of caspase-8, caspase-3, and PARP were distinct after combination treatment. Furthermore, ER stress-related proteins, such as CHOP and GRP78, were activated after combination treatment. We conclude that genistein sensitizes CaSki cells to apoptosis treated with PDT by 9-HpbD-α (0.59 μg/mL) through mechanisms that involve downregulation of the E6*I, E6*II, and E7 oncogenes, activation of caspase-8 or caspase-3, and ER stress.

The Anticancer Effect of Combination of Genistein and Photofrin PDT in Human AMC-HN3 Head and Neck Cancer Cell Lines

Photodynamic therapy (PDT) is a treatment utilizing the generation of singlet oxygen and other reactive oxygen species (ROS), which selectively accumulated in target cells. Genistein, soy-derived phytoestrogen, is one of the anticancer agents found in soybean. In the current study, we investigated the effect of photofrin-induced PDT and genistein on apoptotic cell death in head and neck cell line (AMC-HN3) to confirm the photodynamic therapy of genistein. It was determined by MTT assay that the combination group had more cytotoxicity effect than PDT group alone. Combination of photofrin PDT and genistein induced apoptosis more when comparing with PDT alone. Our data also showed that ROS was increased in combination therapy, indicating apoptosis by mitochondrial damage. These results indicated that the combination of photofrin PDT and genistein showed more cytotoxic effect and induced apoptosis in head and neck cancer cell line.