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近年,癌治療技術の進歩に伴い口腔癌の治療成績も向上してきているが,未だ癌細胞の有する造腫瘍性,浸潤・転移能などの悪性形質に起因する再発,転移により制御不能となる場合も少なくないのが現状である。癌細胞の悪性形質は,必ずしも発癌当初より備わっているわけではなく,主に発癌後の増殖過程において周囲の微小環境との関わり,特に炎症局所において高頻度,長時間持続放出される活性酸素の影響などによって,遺伝子変化が蓄積し獲得されているらしいことが種々の臓器の癌で証明されて来ている。また,口腔癌でも同様の現象が生じている可能性が明らかにされつつあることから,癌細胞の悪性形質獲得の機序解明およびその抑制は口腔癌の研究,治療において重要な今日的課題と考えられる。活性酸素は,本来生体における生理的代謝や各種生化学的反応を行う上で必要不可欠なものであるが,その量的なバランスが崩れた場合には,逆に生体を構成する蛋白質,脂質および核酸などに障害をもたらし様々な疾患を誘発することも知られている。細胞にはmanganese superoxide dismutase (MnSOD), copper and zinc superoxide dismutase (Cu,ZnSOD), gultathione peroxidase (GPx), catalase (CAT)などの抗酸化酵素が存在し活性酸素障害から自らを防御する機構が備わっており,両者間の調節が図られ生体の恒常性が保たれている。従来,これら活性酸素が発癌や癌細胞の悪性化進展に関与する証明は,主として疫学的考察やin vitroにおける抗酸化物質,抗酸化酵素を用いた検討によりなされ,生体内における活性酸素および抗酸化酵素と発癌,悪性化進展との関係を明らかにした研究は少ないのが現状である。また,これらの研究は癌細胞に作用する活性酸素の影響を検討したものが多く,癌細胞側の活性酸素抵抗能,すなわち癌細胞内の抗酸化酵素誘導能の程度と,活性酸素が介在する悪性化進展との関係を検討した研究はない。岡田らは,C57BL/6マウスの線維肉腫BMT-11 cl-9をmutagenであるquercetinで処理して得られたQR癌細胞を用い癌細胞の悪性化進展を再現する動物実験モデルを確立した。すなわち,QR癌細胞を同系正常マウスに単独皮下移植すると自然退縮し腫瘍を形成しないが,ゼラチンスポンジ等の異物と共に皮下移植すると腫瘍を形成する。増殖してきた腫瘍から樹立した培養癌細胞株を新たなマウスに皮下あるいは尾静脈内移植を行うと,もとのQR癌細胞には見られない強い増殖性および肺転移能を示すという悪性化進展モデルを確立した。この悪性化進展を促進する要因として,異物に集積する炎症細胞の産生する活性酸素が関与しているらしいことをin vitro, in vivoの実験で推察している。また,数系のQR癌細胞クローンを用いた実験では,細胞内の抗酸化酵素のうち特にMnSODの蛋白量と悪性化進展を起こす頻度が逆相関することが示されている。すなわち,MnSOD量の少ないQR-32クローン癌細胞は比較的容易に悪性化進展を獲得するが,逆にMnSOD量の多いQR-29クローン癌細胞では悪性化進展に対して抵抗性であることが示されている。そこで,活性酸素がQR癌細胞の悪性化進展の要因であること,および癌細胞内の抗酸化酵素の悪性化進展過程における役割を明らかにするためにMnSOD遺伝子をQR-32癌細胞に導入し過剰発現させ,これがゼラチンスポンジとの同時皮下移植によって起こる悪性化進展に及ぼす影響を検討した。, QR-32 cells are unable to grow in normal C57BL/6 mice when injected s.c. (1×10^5 cells), whereas they grow lethal and are converted to malignant tumor after co-implantation with a foreign body, i.e., gelatin sponge, through mediation of the foreign-body-induced inflammation. Rat manganese superoxide dismutase (MnSOD) cDNA was transfected into a clonal mouse fibrosalcoma cell line, QR-32, to examine suppressive effects of such antioxidative enzyme on inflammatory-cell-mediated tumor progression. MnSOD overexpressing clones (QR-32SOD-23 and 24) were analyzed for their ability to convert the phenotype to malignant tumors in comparison to control vector-transfected cells (QR-32 neo) and wild type QR-32 cells. QR-32SOD-23 cells and QR-32SOD-24 cells had 2.2 and 1.8-fold increases in MnSOD activity, respectively, as compared to QR-32 neo cells. A similar increase was observed in MnSOD immunoreactive protein level by Western blotting using rat MnSOD specific antibody. The effect of MnSOD overexpression on the cell phenotype was determined by co-implantation of the tumor cells with gelatin sponge into C57BL/6 mice. Both QR-32 cells and QR-32 neo cells wich had been co-implanted with gelatin sponge grew progressively in seven out of ten animals. On the other hand, both QR-32SOD-23 cells and QR-32SOD-24 cells grew progressively in two out of ten animals (p

QR-32 cell is low tumorigemc and low metastatic clone derived from 3-methy cholanthrene induced C57BL/6 mouse fibrosarcoma (BMT-11). When QR-32 cells (2×10^5) were sub-cutaneously implanted or 10^6 cells were intravenously injected into normal mice, they all spontaneously regressed. However, when they were subcutaneously co-implanted with gelatin sponge (10×5×3mm), they acquired enhancement of tumorigenicity and metastatic ability and grew lethally. Previous in vitro assays have shown that this malignant progression of QR-32 cells was caused by oxygen radicals released from host cells reactive to gelatin sponge To elucidate the effects of oxygen radicals in vivo, this study examined whether endogeous oxygen radical scavengers induced by bismuth submtrate (BSN) or PSK administration inhibited the malignant progression of QR-32 cells. The QR-32 (2×10^5) cells and gelatin sponge were subcutaneously co-implanted into mice, pre-administered with BSN (5mg/kg/day) or PSK (150mg/kg/day, i p, 1000mg/kg/day p o). The BSN and PSK administration did not affect the tumor growth. The culture lines from these tumors arising in mice were established implanted with QR-32 cells and gelatin sponge, and implanted these culture tumor cells s c or i v into normal mice. Compared with the parental QR-32 cells, the tumor cell lines developed in the normal mice and used for the malignant progression estimation , i e s. c tumorigenicity and i v lung metastasis. All 18 cell lines (100%) obtained from the untreated group showed progress in the malignancy when compared with QR-32 cells However, these cell lines obtained from the BSN administered group indicated malignant progression in only 4 out of 7 cell lines (57.1%), in the same manner cell lines obtained from the PSK (i p) group indicated malignant progression in 3 out of 6 cell lines (50%), for cell lines obtained from PSK (p o ) group indicated in 4 out of 8 cell lines (50%). Endogeous radical scavengers in tumor in mice administered with BSN or PSK were analysed with the immunoblotting and immunohistostaming. These results of the assay showed that the quantity of radical scavengers (metallothionein, Mn-SOD, Cu/Zn-SOD GSH-Px, Catalase) increased over the untreted groups. These findings suggest that oxygen radicals may play an important role in in vivo QR-32 malignancy progress.

author, HIF-1 is involved in tumor progression/metastasis and activated in various cancers. Here we show that HIF-1α, which plays a major role in HIF-1 activation, is over-expressed in preneoplastic hepatocytic lesions from a very early stage during hepatocarcinogenesis in mice and man. Transcriptional targets of HIF-1, such as VEGF, glut-1, c-met and Igf-2, were also over-expressed in mice lesions. HIF-1α expression was independent of hypoxia, because pimonidazole immunostaining was negative in the mouse lesions after treatment with pimonidazole. On the other hand, Akt, the pathway of which can up-regulate HIF-1α expression, was activated in the mouse lesions, while HIF-1α was markedly downregulated in the mouse HCC cell lines after treatment with a PI3 kinase (PI3K) inhibitor, LY294002, indicating that HIF-1α expression is dependent on PI3K/Akt signaling. Conversely, HIF-1α knockdown by siRNA in the HCC cell line resulted in decreased expression of activated Akt together with the HIF-1 target genes, indicating that Akt activation is reversely dependent on HIF-1 activation. Treating the HCC cells with Igf-2 upregulated both phospho-Akt and HIF-1α, while inhibition of Igf-2 by the neutralizing Igf-2 antibody downregulated them both, suggesting that Igf-2 may, at least in part, mediate the activation of Akt and HIF-1α. However, Akt was not activated by Igf-2 or EGF in the HIF-1α knockdown cells, indicating that expression of the HIF-1 target genesis necessary for the Akt activation. These findings suggest that the reciprocal activation of PI3K/Akt signaling and HIF-1α may be important in progression of hepatocarcinogenesis.