Anti-cancer stem cell activity of the Src inhibitor dasatinib in thyroid cancer cells

Although the prognosis of differentiated thyroid cancer (DTC) is good, those of poorly-differentiated and undifferentiated thyroid cancers (PDTC and UDTC) are poor. Recent preclinical studies have suggested that the Src inhibitor dasatinib is active in thyroid cancer cell lines. We conducted the present study in an attempt to clarify the antitumor activity of dasatinib in PDTC and UDTC. The expression levels of c-Src, phosphorylated Srcs (p-SrcY416 and p-SrcY527), focal adhesion kinase (FAK), and phosphorylated FAK (p-FAKY861) were immunohistochemically investigated in a case-control series (15 cases of PDTC or UDTC vs. 29 control cases of DTC). The PDTC cell line KTC-1 and UDTC cell line KTC-2 were used to investigate the anticell growth and anti-cancer stem cell (CSC) activities of dasatinib. The combined effects of dasatinib and the taxane paclitaxel on anti-cell growth and anti-CSC activities were also tested. c-Src and p-FAKY861 expression levels were significantly higher, while those of p-SrcY416 were slightly higher in PDTC and UDTC than in DTC. Dasatinib inhibited cell growth in association with G1-S cell cycle retardation and increased apoptosis in both cell lines. Dasatinib significantly decreased the proportion of CSCs and more than additively enhanced the anti-cell growth activity of paclitaxel. The results of this study suggest that the Src signaling pathway is activated more in PDTC and UDTC than in DTC. The Src inhibitor dasatinib exhibited anti-cell growth and anti-CSC activities. Furthermore, it more than additively enhanced the anti-cell growth activity of paclitaxel

Ethylene Glycol Monomethyl Ether–Induced Toxicity Is Mediated through the Inhibition of Flavoprotein Dehydrogenase Enzyme Family

Ethylene glycol monomethyl ether (EGME) is a widely used industrial solvent known to cause adverse effects to human and other mammals. Organs with high metabolism and rapid cell division, such as testes, are especially sensitive to its actions. In order to gain mechanistic understanding of EGME-induced toxicity, an untargeted metabolomic analysis was performed in rats. Male rats were administrated with EGME at 30 and 100 mg/kg/day. At days 1, 4, and 14, serum, urine, liver, and testes were collected for analysis. Testicular injury was observed at day 14 of the 100 mg/kg/day group only. Nearly 1900 metabolites across the four matrices were profiled using liquid chromatography-mass spectrometry/mass spectrometry and gas chromatography-mass spectrometry. Statistical analysis indicated that the most significant metabolic perturbations initiated from the early time points by EGME were the inhibition of choline oxidation, branched-chain amino acid catabolism, and fatty acid β-oxidation pathways, leading to the accumulation of sarcosine, dimethylglycine, and various carnitine- and glycine-conjugated metabolites. Pathway mapping of these altered metabolites revealed that all the disrupted steps were catalyzed by enzymes in the primary flavoprotein dehydrogenase family, suggesting that inhibition of flavoprotein dehydrogenase–catalyzed reactions may represent the mode of action for EGME-induced toxicity. Similar urinary and serum metabolite signatures are known to be the hallmarks of multiple acyl-coenzyme A dehydrogenase deficiency in humans, a genetic disorder because of defects in primary flavoprotein dehydrogenase reactions. We postulate that disruption of key biochemical pathways utilizing flavoprotein dehydrogenases in conjugation with downstream metabolic perturbations collectively result in the EGME-induced tissue damage

SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER

甲状腺低分化・未分化癌細胞に対するヘッジホッグ阻害薬GANT61の抗腫瘍効果と癌幹細胞制御作用

　甲状腺分化癌は予後良好であるが低分化・未分化癌は予後不良であり，新規治療薬の開発が急務である．多くの悪性腫瘍でヘッジホッグ（Hh）経路の異常な活性化が起こっており，Hh 経路を標的とした治療戦略が有望視されている．Hh 経路の活性化は，腫瘍の生存・増殖・血管新生の促進ばかりでなく，癌幹細胞の制御との関与が示されている．我々は，甲状腺癌細胞を用いてHh経路を阻害するGANT61の抗腫瘍効果並びに癌幹細胞に与える影響を検討した．また，進行甲状腺癌の治療薬として用いられているタキサン系抗癌化学療法薬パクリタキセルとの併用効果も検討した．当教室で樹立された甲状腺低分化癌細胞株KTC-1及び甲状腺未分化細胞株KTC-2，KTC-3を用いてGANT61の細胞増殖，細胞周期，アポトーシス，癌幹細胞比率に与える影響を検討した．また，Hh 経路のeffector であるglioma-associated oncogene （Gli） 1，その下流にある癌幹細胞制御因子（aldehyde dehydrogenase [ALDH], Snail, Slug）や抗アポトーシス分子（survivin,Bcl-2）発現に与えるGANT61の効果を調べた．GANT61は，すべての甲状腺癌細胞株で細胞増殖を用量依存性に抑制した（50% 阻止濃度の平均値: KTC-1細胞は17.2 μM; KTC-2細胞は13.6 μM;KTC-3細胞は13.3 μM）．GANT61は，KTC-1及びKTC-2細胞のsub-G1分画を増加したが，G1-Sブロックは起こさなかった．GANT61は，全ての細胞株において用量依存性にアポトーシス分画を増加し，survivin やBcl-2の発現を低下させた．GANT61は，すべての細胞株でGli1, ALDH, Slugの発現を低下し，癌幹細胞比率を低下させた．以上の結果は，GANT61が甲状腺低分化・未分化癌細胞のsurvivin やBcl-2発現低下を介してアポトーシス誘導し，細胞増殖を抑制し，さらに，Hhシグナル標的因子Gli1, ALDH, Slug の発現低下により癌幹細胞の自己再生能を抑制することを示唆している．さらにGANT61は，すべての甲状腺癌細胞株においてパクリタキセルの細胞増殖抑制効果を増強した．これらの基礎研究の結果は，GANT61が甲状腺低分化・未分化癌の新規治療薬として有望なことを示唆している．　Patients with differentiated thyroid cancer have a good prognosis, but those with poorly-differentiated or undifferentiated thyroid cancer (PDTC or UDTC) do not. Thus, new therapeutics are urgently needed for PDTC and UDTC. As abnormal activation of the hedgehog (Hh) signaling pathway is observed in several malignancies, it is a promising therapeutic target. Activation of the Hh pathway is suggested to promote not only tumor survival, growth and angiogenesis, but also the growth of cancer stem cells (CSC). Therefore, we investigated the anti-cell growth and anti-CSC effects of the Hh inhibitor GANT61 in thyroid cancer cells. In addition, the combined anti-cell growth activity of GANT61 with an anti-thyroid cancer agent, paclitaxel, was evaluated. The effects of GANT61 on cell growth, cell cycle progression, apoptosis and CSC proportion using the Aldefluor and Thyrosphere assays were measured in the KTC-1 PDTC cell line, and KTC-2 and KTC-3 UDTC cell lines. All cell lines were established at our institute. We also examined the influence of GANT61 on the expression levels of the Hh effector glioma-associated oncogene (Gli) 1, its down-stream CSC-related molecules, aldehyde dehydrogenase (ALDH), Snail and Slug, and anti-apoptotic molecules, Bcl-2 and survivin. GANT61 dose-dependently inhibited the growth of all cell lines (mean 50% inhibitory concentrations: 17.2 μM for KTC-1 cells, 13.6 μM for KTC-2 cells and 13.3 μM for KTC-3 cells) in association with increased apoptosis, and decreased expression of survivin and Bcl-2. Furthermore, the proportion of surviving CSC cells decreased with decreased expression of Gli1, ALDH and Slug. These results demonstrate that GANT61 induced apoptosis via decreased expression levels of survivin and Bcl-2, and inhibited cell growth in thyroid cancer cells. Moreover, GANT61 reduced the expression levels of Hh target genes, such as Gli1, ALDH and Slug, and inhibited CSC self-renewal. GANT61 also enhanced the anti-cell growth effects of paclitaxel in all three thyroid cancer cell lines. Therefore, GANT61 may be a promising antitumor therapy for patients with PDTC or UTC

Exercise and functional foods

Appropriate nutrition is an essential prerequisite for effective improvement of athletic performance, conditioning, recovery from fatigue after exercise, and avoidance of injury. Nutritional supplements containing carbohydrates, proteins, vitamins, and minerals have been widely used in various sporting fields to provide a boost to the recommended daily allowance. In addition, several natural food components have been found to show physiological effects, and some of them are considered to be useful for promoting exercise performance or for prevention of injury. However, these foods should only be used when there is clear scientific evidence and with understanding of the physiological changes caused by exercise. This article describes various "functional foods" that have been reported to be effective for improving exercise performance or health promotion, along with the relevant physiological changes that occur during exercise

転移・再発乳癌患者に対するエリブリン療法の有用性

　エリブリンはタキサンとは異なる作用機序をもつ微小管阻害剤である．海外の第Ⅲ相試験 では，エリブリンの転移・再発乳癌に対する延命効果が示されている．今回，エリブリンの臨床 的な有用性を検討するため，2011年９月から2017年８月に当科でエリブリン療法を行った進行・ 再発乳癌97症例を対象として後方視的に調査した．対象患者の年齢は35 － 81歳（中央値58）， performance status は１が最多で64例，Stage Ⅳが５例，再発が92例であった．原発腫瘍のエス トロゲン受容体は陽性が64例，プロゲステロン受容体は陽性が48例，human epidermal growth factor receptor 2は陰性が78例であった．前化学療法のレジメン数は0 － 9（中央値２）, 臓器転 移ありが69例，肝転移ありが40例，エリブリン療法の実施サイクル数は1 － 12回（中央値3.5）， 観察期間は1 － 55か月（中央値10），有害事象による中止例は10例であった．最大治療効果は，完 全奏効が０例，部分奏効が１例，長期安定が27例，安定が16例，進行が42例，不明・評価不能が 11例であった． 臨床的有効率（奏効率＋長期安定率）は29% であった．Time-to-treatment failure (TTF) は0 － 178週間（中央値13），治療開始後全生存期間は0 － 55か月（中央値15.5）であった． 好中球減少症はグレード１が最多で61例，非血液毒性は嘔気が７例，肝機能障害が６例，末梢神 経障害が５例，間質性肺炎が3例などであった．良好なTTF の予測因子は，単変量解析で「臓器転 移なし」（P = 0.0356）が同定された．良好な治療開始後生存の予測因子は，多変量解析にて「臨 床的有効性あり」（P = 0.0008）と「PS が０か１」（P < 0.0001）が同定された．エリブリン療法は， 奏効率は低かったが，本療法は，約30% の症例に臨床的有効性をもたらし，生存期間の延長に寄 与する可能性がある．　Eribulin is an anti-microtubule agent that uses a different mechanism of action to taxanes. Phase 3 clinical trials have shown that eribulin exerts life-prolonging effects in patients with metastatic or recurrent breast cancer. In order to investigate the utility of eribulin, we conducted a retrospective and observational study on 97 patients with metastatic and recurrent breast cancer who were treated in our institute between September 2011 and August 2017. The median age of the patients was 58 years (range: 35 － 81). The performance status was 1 in 64 patients. Five patients had stage IV disease, while 92 had recurrent disease. Sixtyfour patients had estrogen receptor-positive tumors, 48 had progesterone receptor-positive tumors, and 78 had human epidermal growth factor receptor 2-negative tumors. The median number of regimens of previous chemotherapies was 2 (range: 0 － 9). Sixty-nine patients had visceral metastases, while 40 had liver metastases. The median number of cycles of eribulin therapy was 3.5 (range: 1 － 12). The median follow-up period was 10 months (range: 1 － 55). The best responses to therapy were a complete response in 0 patients, a partial response in 1, long-term stable disease in 27, stable disease in 16, progressive disease in 42, and unevaluable in 11. The clinical benefit rate (objective response rate + long-term stable disease rate) was 32%. The median time-to-treatment failure (TTF) was 13 weeks (range: 0 － 178). Median overall survival (OS) after the initiation of therapy was 15.5 months (range 0 － 55). The most frequent grade of neutropenia was 1, which was observed in 61 patients. Major non-hematological toxicities were nausea in 7 patients, liver dysfunction in 6, peripheral neuropathy in 5, and interstitial pneumonitis in 3. Univariate and multivariate analyses identified “no visceral metastasis” as the only predictive factor for TTF (P = 0.0356 for the multivariate analysis). The multivariate analysis revealed that“ the presence of a clinical benefit by the therapy” (P = 0.0008) and “a performance status of 0 or 1” (P < 0.0001) were independent predictive factors for OS. Eribulin therapy for patients with metastatic or recurrent diseases provided clinical benefits for approximately 30% of patients. These results suggest that this therapy prolongs the OS of patients

進行・再発乳癌患者に対するフルベストラントの有用性

　ホルモン受容体陽性の閉経後進行・再発乳癌患者に対する内分泌療法として，フルベストラントが本邦で臨床導入されて６年余りが経過した．本剤の有用性を検討するため，2012年１月〜2016年10月に川崎医科大学附属病院乳腺甲状腺外科において，フルベストラントが単独使用され，治療評価が可能であった51症例の電子カルテを後方視的に調査した．対象患者の年齢の中央値は70歳．進行例が９例，再発例が42例．臓器転移ありが23例．観察期間の中央値は18か月．前内分泌療法数の中央値は２．前化学療法歴ありは21例．治療効果は，完全奏効が３例，部分奏効が６例，安定が25 例（うち長期安定は20例），進行が16例であった．客観的奏効は９例（17.6%），臨床的有用は29例（56.9%）であった．無増悪生存（PFS）期間の中央値は８か月，全生存（OS）期間の中央値は34か月であった．治療効果の予測因子を調べるため，サブグループに分けてPFS及びOS を解析した．肝転移の有無では，PFS 期間の中央値は，なしが9.5か月，ありが５か月（P= 0.0386），OS 期間の中央値は，なしが41か月，ありが15か月（P = 0.0036）であった．前化学療法の有無では，PFS 期間の中央値は，なしが12.5か月，ありが3.5か月（P < 0.0001），OS 期間の中央値は，なしが41か月，ありが24か月（P = 0.0208）．多変量解析では，前化学療法歴の有無が唯一のPFS の有意な予測因子であった．また，肝転移の有無が唯一のOS の有意な予測因子であった．有害事象は６例（11.7%）に認めたが，いずれも軽微であり治療が中断されることはなかった．要約すると，ホルモン受容体陽性の進行・再発乳癌の治療薬として，フルベストラントは17.6% の客観的奏効率，56.9% の臨床的有用性が認められ，既知の報告と同等の治療効果であった．フルベストラントは，化学療法歴のある症例，肝転移のある症例では，有用性が低いと考えられた．　Fulvestrant has been used for the treatment of postmenopausal patients with advanced or recurrent breast cancer in Japan for over six years. To investigate the utility of fulvestrant, we retrospectively reviewed electronic medical records and evaluated the responses of 51 patients with advanced or recurrent breast cancer treated with fulvestrant alone at the Department of Breast and Thyroid Surgery, Kawasaki Medical School Hospital between January 2001 and December 2016. The median age of the subjects was 70 years old. Nine had stage IV diseases and 42 had recurrent diseases. Twenty-three patients had visceral metastases. The median follow-up time after the start of fulvestrant treatment was 18 months. The median number of previous endocrine therapies was 2. Twenty-one patients received chemotherapy previously. Three patients had a complete response, six had a partial response, 25 had a stable disease including 20 patients with a long-term stable disease, and 16 had progressive disease. The objective response rate was 17.6% (9 out of 51), and the clinical benefit rate was 56.9% (29 out of 51). The median progression-free survival (PFS) time was 8 months. The median overall survival (OS) time was 34 months. To investigate predictive factors for response to fulvestrant, subgroup analyses were performed. For liver metastasis, the median PFS and OS time were 9.5 and 41.0 months, respectively, in patients without liver metastasis but 5.0 and 15.0 months, respectively, in those with liver metastasis (P = 0.0386 and P = 0.0036, respectively). For previous chemotherapy, the median PFS and OS time were 12.5 and 41.0 months, respectively, in patients without previous chemotherapy but 3.5 and 24.0 months, respectively, in those with previous chemotherapy (P < 0.0001 and P = 0.0208, respectively). In addition, the Cox’s proportional hazards model revealed that previous chemotherapy was only an independent predictive factor for PFS and that liver metastasis was only an independent predictive factor for worse OS. Although toxicities were recorded in 6 of 51 patients (11.7%), all instances were slight and no patient stopped fulvestrant therapy because of toxicities. In summary, fulvestrant therapy at our hospital provided a 17.6% objective response rate and 56.9% clinical benefit rate in patients with advanced or recurrent breast cancer. These results were similar to those reported previously. According to our subgroup analyses, fulvestrant was unlikely to be effective in patients who received previous chemotherapy or had liver metastasis