Oleoyl triterpene glycoside biotransformed from ginseng suppresses growth and metastasis of murine B16-F10 melanoma via immunostimulation

The effects of 20 (S) -protopanaxadiol 20-O-β-D-glucopyranoside (M1) and 3-O-Oleoyl M1 (OM1) on the growth and metastasis of murine B16-F10 melanoma cells were examined in C57BL/6 mice. A single co-injection of M1 (5 mg/kg) with B16-F10 cells into the liver inhibited tumor growth at the inoculation site by 23 % (not significant compared to untreated control) . In contrast, the same dosage of OMI caused a 2.6-fold suppression of tumor growth, compared with M1 treatment (p <0.02). Concerning the pharmacokinetics, both M1 and OM1 were selectively taken up into the liver soon after i.v. administration (30 mg/kg) . Thereafter, M1 was cleared immediately from the liver ; however, OM1 was retained in the liver at a level of more than 25 % of the administered dose for 24 h after administration. Thus, the antitumor activity paralleled the pharmacokinetic behavior. Moreover, three consecutive i.v. administrations of OM1(30 mg/kg) inhibited the liver metastasis produced by intrasplenic inoculation of B16-F10 cells by 95 %. OM1 did not directly affect tumor growth in vitro, whereas it promoted tumor cell lysis by lymphocytes, particularly non-adherent splenocytes, in a concentration-dependent manner. These results suggest that fatty acid esterification of M1 potentiates the antitumor activity of the parental M1 through delay of the clearance and through immunostimulation. 薬用人参の主要成分であるginsenosideは,腸内細菌によって活性型に加水分解される。20(S)-Protopanaxadiol 20-O-β-D-glucopyranoside(M1)は,protopanaxadiol系サポニンの主要な代謝物である。すでに,我々はM1が脂肪酸とエステル化されることを示してきた。本研究では,C57BL/6マウスを用いてM1及ぴ3-O-oleoyl M1(OM1)の薬物動態と抗腫瘍活性を調べた。これらの薬物は,静脈内投与(30mg/kg)後直ちに肝臓に選択的に取り込まれた。その後,Mlは肝臓から速やかに除去されたが,OM1は投与後24時間,投与量の25%以上のレベルで肝臓に残留した。OM1の脱アシル化によって生じるM1は,投与後6時間まで僅かに検出され,脱アシル化率が非常に低いことが示ざれた。薬物勤態は,抗腫瘍活性に反映した。Ml(5mg/kg)をマウスメラノーマB16-F10細胞と共に肝臓に注入すると,移植部位での腫瘍増殖は23%抑制されたが(非投与対照との有意差ない,OM1処置はM1処置に比べ2.6倍の抑制を示した(p<0.02)。さらに,癌細胞の脾内移植後OM1を3回連続して静脈内投与(30mg/kg)した結果,肝転移を95%抑制した。OM1は in vitro の腫瘍増殖に直接影響を及ぼさなかったが,リンパ球,持に非接着性細胞による腫瘍傷害活性を濃度依存的に増強した。以上の結果から,OM1は生体内クリアランスの遅延及ぴ抗腫瘍免疫の誘導を通してMlの抗腫瘍効果を増強することが示唆された

Organ selectivity of Juzen-taiho-to and Ninjin-yoei-to in the expression of anti-metastatic efficacy

本研究では,マウス結腸癌Colon26-L5細胞の門脈内移入により形成される肝転移および同細胞の尾静脈内移入により形成される肺転移に対する,十全大補湯および人参養栄湯の経口投与による抑制効果を検討した。十全大補湯は肝転移に対して有意に抑制したが,肺転移には抑制効果を示さなかった。これに対して,人参養栄湯は逆の効果,すなわち肝転移には効果を示さなかったが,肺転移に対して有意な抑制を示した。このように,同一の癌細胞と同系のマウスの転移病態モデルを用いて,二つの方剤の臓器選択的な転移抑制効果が観察された。このような効果発現の差異の解釈は,13世紀に確立されたtheory of Jingand Lun(引経報使)の考えに,部分的に通ずるものがあると思われる。 We investigated the inhibitory effect of oral administration of Juzen-taiho-to and Ninjin-yoei-to on liver metastasis caused by intraportal vein injection of colon 26-L5 cells and lung metastasis by intravenous injection of same tumor cells. Juzen-taiho-to significantly inhibited liver metastasis but not lung metastasis. In contrast, Ninjin-yoei-to was effective at inhibiting lung metastasis but not liver metastasis. In the experimental liver and lung metastases model using same tumor in syngeneic mice system, oral administration of both formulations showed a differential pattern with organ selectivity for the expression of anti-metastatic effects. These results suggest that the different expression of the anti-metastatic effects of both Kampo medicines on tumor metastasis are partly based on the medicinal guides according to the theory of Jing and Lun (Inkei-hoshi) formed in the 13th century

Selective anti-cancer activity of Hirsutine against HER2 positive breast cancer cells by inducing DNA damage

Hirsutine is one of the major alkaloids isolated from plants of the Uncaria genus and is known for its cardioprotective, anti‑hypertensive and anti-arrhythmic activities. We recently reported that hirsutine is an anti-metastatic phytochemical by targeting NF-κB activation in a murine breast cancer model. In the present study, we further examined the clinical utility of hirsutine against human breast cancer. Among six distinct human breast cancer cell lines, hirsutine showed strong cytotoxicity against HER2-positive/ p53-mutated MDA-MB‑453 and BT474 cell lines. Conversely, HER2-negative/p53 wild‑type MCF-7 and ZR-75-1 cell lines showed resistance against hirsutine-induced cytotoxicity. Hirsutine induced apoptotic cell death in the MDA-MB-453 cells, but not in the MCF-7 cells, through activation of caspases. Furthermore, hirsutine induced the DNA damage response in the MDA-MB-453 cells, but not in the MCF-7 cells, as highlighted by the upregulation of γH2AX expression. Along with the induction of the DNA damage response, the suppression of HER2, NF-κB and Akt pathways and the activation of the p38 MAPK pathway in the MDA-MB-453 cells were observed. Considering that there was no difference between MDA-MB-453 and MCF-7 cells in regards to irinotecan‑induced DNA damage response, our present results indicate the selective anticancer activity of hirsutine in HER2-positive breast cancer by inducing a DNA damage response

Induction of apoptosis in Lewis lung carcinoma cells by an intestinal bacterial metabolite produced from orally administered ginseng protopanaxadiol saponins

The present study demonstrated that oral administration of an intestinal bacterial metabolite (M1) of protopanaxadiol-type saponin significantly inhibited the tumor growth at the implantation site after intrapulmonary implantation of Lewis lung carcinoma (LLC) cells, and suppressed the metastasis to mediastinal lymph nodes. We also investigated the inhibitory mechanism of M1 on the growth of LLC cells. M1 inhibited the proliferation of LLC cells in a concentration-dependent manner, with characteristic morphological changes at the concentration of 30 μM. Treatment of LLC cells with M1 resulted in marked elevation of the caspase-3 activity, peaking at 2 h, and a subsequent time-dependent induction of apoptosis during the period from 3 to 24 h, as evidenced by DNA fragmentation analysis. Since M1-induced growth inhibition of LLC cells was completely abrogated by the pretreatment with a specific inhibitor of caspase-3, Z-DEVD-FMK, M1 functions via the activation of caspase-3 in the process of apoptosis in LLC cells. Thus, the anti-proliferative activity of M1 against LLC cells is primarily due to the induction of apoptosis via promotion of caspase-3 activity, and this induction may lead to the anti-tumor activity in vivo

Anti-invasive effects of curcuminoid compounds from Curcuma aromatica Salisb. on murine colon 26-L5 carcinoma cells

ショウガ科に属する Curcuma aromatica Salisb. の根茎のクロロホルム抽出エキスから,化学構造の明らかな4種のクルクミンおよびその関連化合物:curcumin(CA-1),demethoxycurcumin(CA-2),5\u27-methoxycurcumin(CA-3),bisdemethoxycurcumin(CA-4)を分離した。これらの化合物を用いてマウス結腸癌細胞(colon 26-L5)に対する増殖,基底膜への浸潤,細胞運動に及ぼす効果について検討した。クルクミン(CA-1)とその関連化合物(CA-2,3 および4)は,細胞に対して傷害性を示さない10μMの濃度において,マウス結腸癌細胞の基底膜への浸潤を抑制した(それぞれ22.8,28.9,10.3および62.0%の抑制率)。この癌細胞の運動能に対しても同様の抑制効果が観察された。これらのクルクミン関連化合物の中で,CA-4は強い抑制活性を持ち,癌細胞の浸潤および運動能に対して濃度依存的な抑制効果を示した。このように,クルクミン関連化合物の芳香族環のhydroxyl基およびmethoxyl基が癌細胞の浸潤活性の発現と関係している可能性が示唆された。 Bioassay-directed fractionation of the active chloroform extract from the rhizomes of Curcuma aromatica Salisb. (Zingiberaceae) led to the isolation of four main curcuminoid constituents: curcumin (CA-1), demethoxycurcumin (CA-2), 5\u27-methoxycurcumin (CA-3) and bisdemethoxycurcumin (CA-4). This is the first report to describe the isolation of CA-3 from C. aromatica. The chemical structures of these compounds were determined on the basis of spectral analysis and their inhibitory effects on the proliferation, invasion and migration of murine colon 26-L5 adenocarcinoma cells were evaluated in vitro. Curcumin and its analogues (CA-2, 3 and 4), at the non-cytotoxic concentration of 10μM, inhibited the invasive ability of colon 26-L5 cells to the ranges of 22.8, 28.9, 10.3 and 62.0%, respectively. A similar effect of these constituents on the migration of colon 26-L5 cells was also observed. Among these curcuminoids, CA-4 showed the strongest activities, inhibiting both tumor cell invasion and migration in a concentration-dependent manner

An intestinal bacterial metabolite (M1) of ginseng protopanaxadiol saponins inhibits tumor-induced neovascularization

The present study demonstrated that an intestinal bacterial metabolite (M1) of protopanaxadiol-type ginsenosides significantly inhibited the growth of implanted tumor and the intrahepatic metastasis by the implantation of a small fragment of colon 26-L5 tumor into the liver when it was administered orally. These findings indicates that M1 was effective for the inhibition of the growth and metastasis of colon26-L5 cells in addition to lung metastasis of B16-BL6 melanoma cells as have been reported previously. The conditioned medium of colon 26-L5 cellS (CM-L5) induced in vitro tube formation of hepatic sinusoidal endothelial (HSE) cells on Matrigel-coated substrates, which is considered to be an important step in the processes of tumor angiogenesis. \u27This activity of CML5 was abrogated by noncytotoxic concentrations of M1 in a concentration-dependent manner. Similarly, M1 eliminated the ability of CM-L5 to promote the migration of HSE cells concentration-dependently. These findings indicate that M1-induced inhibition of tumor growth and intrahepatic metastasis may be partly related to the suppression of tumor angiogenic responses including capillary tube formation and migration of HSE cells. 本研究では,薬用人参(Panax ginseng C.A.MEYER)のprotopanaxadiol-type ginsenosidesの主要な腸内細菌代謝物M1の経口投与は,マウス結腸癌colon 26-L5細胞の腫瘍小片を肝へ直接移植した後の移植部位での増殖と肝内移転に対して有意に抑制効果を示すことを明らかにした。この結果は,B16-BL6メラノーマ細胞による肺転移を抑制した以前の報告と同様に,co1on 26-L5細胞に対しても有効であることが示された。肝類洞内度細胞(HSE細胞)をマトリゲルをコートした基質上で,colon 26-L5細胞の培養上清(CM-L5)とともに培養すると,腫瘍血管新生の週程における重要なステップのひとつである,内度細胞の菅腔形成を誘導した。CM-L5による菅形成能は,細胞傷害性を示さない濃度範囲のM1により,濃度依存的に抑制された。同様に,CM-L5によるHSE細胞の移動能の亢進を,M1は濃度に依存して抑制した。以上,M1による結腸癌の肝における増殖及び肝内転移の抑制は,内度細胞の骨腔形成及ぴ移動能を含む血管新生反応の抑制と部分的に関係していることが示唆された

Immunopharmacological properties of Oren-gedoku-to (a Kampo medicine, Huang-Lian-Jie-Du-Tang) on contact hypersensitivity reaction in mice

We investigated the effects of Oren-gedoku-to (Huang-Lian-Jie-Du-Tang), a Kampo medicine, on DNFB-induced contact hypersensitivity (CHS) response in mice in order to further clarify the immunopharmacological properties of this formulation. 1) Administration of Oren-gedoku-to decreased the magnitude of ear swelling in the CHS response and shortened the affected period. The inhibitory effect on ear swelling was observed even when Oren-gedoku-to was given orally with different timing schedules. 2) The expressions of mRNAs for CD8, IFN-7 and TNF-α in the ear of Oren-gedoku-to-treated mice were markedly decreased 24 h after the challenge. 3) The number of skin-draining regional lymph node cells (LNCs), CD4^+ T cells and CD8^+ T cells was decreased without affecting the ratio of CD8^+/CD4^+ T cells. 4) Oren-gedoku-to resulted in a marked impairment of the hapten-specific development of LNCs. These results suggest that the suppressive effect of Oren-gedoku-to on ear swelling was partly caused by the suppression of lymphocyte proliferation. 接触過敏反応(CHS)に対する黄連解毒湯の抑制効果について検討した。1g/kgの黄連解毒湯を感作日より連続投与することで,DNFB塗布による耳介の腫脹は軽減し,その持続時間も短縮した。また,黄連解毒湯の投与期間を変更(感作後0-2日間あるいは4-6日間の投与)しても抑制効果が認められた。耳介局所では,黄連解毒湯の連続投与により,CD8,IFN-γおよびTNF-αのmRNA発現は減弱した。所属リンパ節では,全リンパ節細胞,CD8^+T細胞,CD4^+T細胞の数が減少したが,CD8/CD4比に変化はみられなかった。さらに,リンパ節細胞のハプテン特異的な増殖能は抑制された。以上の結果より,黄連解毒湯のCHSの抑制効果にハプテン特異的リンパ球の増殖抑制が関与していると考えられた

Mammary tissue microenvironment determines T cell-dependent breast cancer-associated inflammation

Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis-prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis-prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor-jb (NF-jB) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF-jB ⁄ Luc2 reporter gene (4T1 NF-jB cells), we observed significantly increased bioluminescence approximately 7 days after metastasis-prone orthotopic mammary fat-pad inoculation but not ectopic s.c. inoculation of 4T1 NF-jB cells. Such in vivo NF-jB activation within the fat-pad 4T1 tumor was diminished in immune-deficient SCID or nude mice, or T celldepleted mice, suggesting the requirement of host T cell-mediated immune responses. Given the fat-pad 4T1 tumor expressed higher inflammatory mediators in a T cell-dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells

Effect of some Kampo medicines, including Tokaku-joki-to (Tao-He-Cheng-Qi-Tang), on IgE-mediated triphasic skin reaction in passively sensitized mice

Previous studies have reported that the mice passively sensitized with anti-DNP IgE antibody exhibited IgE-mediated biphasic cutaneous reaction with an immediate phase response (IPR) at 1 h and a late phase response (LPR) at 24 h after the challenge of DNFB (dinitrofluorobenzene) . We recently found that the third phase inflammatory response with intense and persisting infiltration of eosinophils, named very late phase response (vLPR) , was induced following IPR and LPR in response to DNFB in passively sensitized mice, and that the peak response of vLPR was on the 8^ day after the challenge. The inhibitory effect of Kampo medicines on the triphasic cutaneous inflammatory reaction was divided into several groups in terms of their inhibition rate of ear swelling. Among the formulations, Tokaku-joki-to (Tao-He-Cheng-Qi-Tang) was effective at inhibiting IPR, LPR and vLPR ( +/+/+ group) and scratching behavior in IPR. The Inhibitory effect of Tokaku-joki-to on triphasic cutaneous reaction primarily depends on its composed crude drugs, Glycyrrhizae Radix and Cinnamomi Cortex. These findings indicate that Tokaku-joki-to formulation is usefull for the inhibition of cutaneous inflammatory diseases. 抗DNP IgE抗体で受動感作したマウスの耳介にDNFB(ジニトロフルオロベンゼン)を塗布することにより,1時問およぴ24時間目をピークとする即時相反応(IPR)およぴ遅発相反応(LPR)からなるIgE介在二相性皮膚反応を示すことがすでに知られている。我々は最近,この受動感作マウスにおいてDNFBによる反応惹起後にIPR,LPRに続く,三相目の強い炎症性反応を見出し,超遅発相反応(vLPR)と名付けた。これは抗原塗布から8日目をピークとする,著明かつ持続的な好酸球の浸潤を伴う腫脹反応である。種々の漢方方剤を用いて,この三相性皮膚反応に対する抑制効果を検討した結果,各相の耳介腫脹の抑制率に基づき,いくつかのグループに分類された。検討した方剤中,桃核承気湯はIPR,LPR,vLPRの三相反応に対して抑制を示し(+/+/+群),さらにIPRで観察される耳介の掻き行動(痒みの指標と考えられる)を抑制した。桃核承気湯の三相性皮膚反応に対する抑制効果の発現は,主として構成生薬である甘草およぴ桂皮に基づくことが示唆された。これらの知見から,漢方方剤:桃核承気湯が炎症性皮膚疾患に有効であることが示された