Cancer chemopreventive potential of natural product-derived stilbenoids in multistage carcinogenesis

Abstract

Cancer is the major health burden in the world despite of notable advances in diagnosis and treatment of cancer in past decades. Carcinogenesis is a multistage process of cancer formation, and generally takes place through consecutive three stages of initiation, promotion and progression. It is estimated that the amount of time for the transformation of normal cells to malignant cells is from several years to decades. This rather long latent period in developing cancer might provide a good rationale for chemoprevention before malignancy. Cancer chemoprevention is defined as the preventing, delaying, or reversing in the process of carcinogenesis using nontoxic synthetic compounds or natural substances before malignancy. In recent years, many researchers have focused on addressing the chemopreventive agents from natural products. Also, it has been reported that resveratrol, a naturally occurring stilbenoid, might be one of candidates of cancer chemopreventive agents. In addition, in the previous studies, pinosylvin and its synthetic derivatives, structurally similar to resveratrol, exerted various chemopreventive activities in vitro assay systems. One of pinosylvin derivatives has been shown the induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) activity. Also, pinosylvin and the derivative exerted anti-inflammatory activities via the downregulation of both the production of prostaglandin E₂ (PGE₂)and nitric oxide (NO). Moreover, pinosylvin and its derivatives exhibited anti-proliferative activities on various human cancer cell lines. Finally, pinosylvin and its derivatives demonstrated anti-metastatic potential in HT1080 cells. On the basis of previous findings, in this study, the cancer chemopreventive activities and underlying mechanisms of pinosylvin and the active derivatives were examined by use of in vitro and in vivo model systems. At first, the effect of 3,5-dimethoxy-trans-stilbene (DMS), an active compound selected by NQO assay system, on the induction of the detoxifying enzymes and the underlying mechanism of action were evaluated. DMS increased NQO1 activity in accordance with the upregulation of both protein and mRNA expressions of NQO1. The induction of NQO1 transcription was in part due to the nuclear translocation and activation of nuclear factor E2-related factor 2 (Nrf2) modulated by the upstream signal transduction molecule, protein kinase C δ (PKCδ). In summary, these data suggested that DMS could be a possible cancer chemopreventive agent by inducing detoxifying enzymes. It has been reported that chronic inflammation might be involved in the development of various cancers. Inflammatory proteins such as COX-2 and iNOS are intimately related to the promotion process of carcinogenesis. In the previous study, pinosylvin and its derivatives showed inhibitory effects on the production of inflammatory mediator PGE2 in lipopolysaccharide (LPS)-stimulated RAW 264.7 mouse macrophage cells. Especially, 3-hydroxy-5-(2-ethyl-pentanoyl)-trans-stilbene (HEPTS) exhibited the potent inhibitory effect on the production of NO as well as PGE2 among tested compounds. Therefore, the mechanisms underlying anti-inflammatory activities of HEPTS were examined. As a result, HEPTS exhibited the inhibitory effects on the expression of the inducible iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. The modulation of TIR-containing adapter molecule (TRIF)-dependent JAK/STAT1 pathway might be involved in the downregulation of iNOS expression by HEPTS. In addition, pinosylvin and HEPTS exhibited anti-promotion activities in DMBA-initiated and TPA-promoted two-stage mouse skin carcinogenesis model with decreasing promotion-related biomarkers. Taken together, these data suggested that pinosylvin and HEPTS could be possible cancer chemopreventive agents acting on the tumor promotion stage with anti-inflammatory properties. Pinosylvin and HEPTS pronouncedly exhibited anti-proliferative activities against various cancer cells among pinosylvin and its derivatives. On the basis of these findings, anti-proliferative activities and underlying mechanisms of pinosylvin and its derivative HEPTS were investigated in HCT 116 human colorectal carcinoma cells. First, treatment with 60 μM of pinosylvin exerted the anti-proliferative activity by arresting transition of cell cycle from G1 phase to S phase in HCT 116 cells in accordance with the downregulation of the protein levels including c-myc, anti-proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin E, cyclin A, cyclin dependent kinase 4 (CDK4) and CDK2 as well as serine phosphorylation in pRb. Also, pinosylvin increased the protein expression of CDK inhibitor p21, and tumor suppressor p53. The effect of pinosylvin was due to the inhibition of Akt activation which gave rise to inhibit several downstream signaling cascades including forkhead transcription factor in rhabdomyosarcoma like-1 (FKHRL1), mammalian target of rapamycin (mTOR), and glycogen synthase kinase 3 β (GSK-3β). Also, pinosylvin inhibited the translocation of β-catenin concomitant with the downregulation of mRNA expression of β-catenin target genes. Moreover, analysis of the gene expression profiled by cDNA microarray showed that pinosylvin regulated gene expressions related to cell proliferation such as GDF15, CDKN1A, TSC22D1, PCNA, CDC2, CDK8, HSPCB, PTGES2, CDK2AP1, CKS2, and CDCA2 in HCT 116 cells. Pinosylvin also suppressed the protein expression of COX-2 in specific HCT 116 cells which were stably transfected with COX-2-encoding plasmid. In addition, HEPTS exhibited a significant anti-proliferative activity with an accumulation of cells in G0/G1 and sub-G1 phases in HCT 116 cells. HEPTS suppressed the expression of G1 phase-regulatory proteins including cyclin D1, cyclin A, cyclin E, and CDK2, and the phosphorylation of pRb. Moreover, HEPTS exerted suppressive effects on the active phosphorylation of mitogenic proteins including Src, signal transducer and activator of transcription 3 (STAT3), and extracellular signal-regulated kinase 1/2 (ERK1/2). Also, HEPTS caused the suppressive effects on the phosphorylation of phosphoinositide-dependent protein kinase 1 (PDK1) at Ser241 residue, Akt at Ser473 residue, GSK-3β at Ser9 reside in a dose-dependent manner. Furthermore, HEPTS inhibited the nuclear translocation of β-catenin, resulting in the decreased mRNA levels of β-catenin target genes. In addition, the mRNA expression of E-cadherins which negatively regulate β-catenin was upregulated by HEPTS. These data, taken together, suggested that pinosylvin and HEPTS might be possible candidates for cancer chemopreventive agents by exerting anti-proliferative activities with various mechanisms. Metastasis is the migration of malignant cancer cells from the primary neoplasm to distant secondary organs or tissues. In the preliminary study, pinosylvin and DMS exhibited the inhibitory effects on the mRNA expression of metalloproteinase-2 (MMP-2), wound healing and colony dispersion among test compounds. On the basis of these findings, anti-metastatic activities and underlying mechanisms of pinosylvin and DMS were investigated in HT1080 human fibrosarcoma cells. Pinosylvin and DMS downregulated the mRNA expression of MMP-2, MMP-9, membrane type 1-MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 1 (TIMP-1), whereas these compounds did not affect the mRNA expression of TIMP-2. The suppressive effect of DMS on the mRNA expression of MMP-9 was coincided with the inhibition of protein expression and enzymatic activities of MMP-9 in gelatin zymography. To further elucidate the molecular mechanism of DMS on the downregulation of MMP-9, the effects of DMS on the major transcription factor of MMP-9 including AP-1 and NF-κB were examined. DMS caused the inhibition in AP-1 transcriptional activity, while not affecting NF-κB transactivation. This was accompanied by the suppression of ERK1/2 by inhibiting phosphorylation at Thr202 and Tyr204 residues. To better understand the effects of pinosylvin and DMS on cancer metastasis, pinosylvin and DMS were applied to in vivo pulmonary metastasis model system. Both pinosylvin and DMS decreased the formation of nodules with the downregulation of MMP-2 and MMP-9 enzymatic activities as well as COX-2 expression in lung tumors. These findings demonstrated that pinosylvin and its derivatives DMS might be potential candidates for anti-metastatic agents. All taken together, these data suggested that natural product-derived stilbenoid pinosylvin and its derivatives DMS and HEPTS might be potential candidates for cancer chemopreventive agents with multiple mechanisms of actions.;암은 전세계적으로 흔히 발생하는 질병 중의 하나로서, 최근 진단법 및 치료 기술의 발전에도 불구하고 암발생률 및 암사망률은 크게 감소하지 않은 것으로 나타났다. 따라서, 이미 생성된 암을 효과적으로 치료하는 방법을 개발하는 동시에, 적극적으로 암을 예방하고 암화를 저해하는 화학적 암예방이 필요하다. 화학적 암예방은 독성이 없는 천연 유래 물질 및 합성물을 섭취하여, 암의 발생 및 암화 과정을 억제, 지연, 혹은 역행시키는 방법으로, 현재 resveratrol 등의 천연 유래 물질에 대한 암예방 활성 연구가 활발히 진행 중이다. 이러한 배경에 따라, 본 연구에서는 resveratrol과 구조적 유사성을 지닌 천연 stilbenoid인 pinosylvin 및 그 유도체에 대하여 암예방 활성을 평가하였고, 효능을 지닌 물질에 대한 작용 기전을 규명하였다. 우선, 본 연구에서는 정상세포에 변이를 유발하는 발암물질을 해독화하는 물질을 발굴하고 그 작용기전을 연구하였다. NAD(P)H:quinone oxidoreductase 1 (NQO1) 효소 활성 실험을 통하여 pinosylvin 유도체인 3,5-dimethoxy-trans-stilbene (DMS)의 NQO1 효소 활성 유도 작용을 확인하였으며, 이후 NQO1 단백질 및 mRNA 발현 증가를 확인하였다. 이러한 DMS의 활성은 NQO1의 발현을 조절하는 중요한 전사조절인자인 (nuclear factor E2-related factor 2) Nrf2의 핵내 이동 증가 및 NQO1 전사 활성 증가에 기인하는 것으로 여겨진다. 또한, DMS에 의한 Nrf2의 활성화는 일부 protein kinase C δ (PKCδ) 작용에 기인하는 것으로 추정된다. 또한, 기존 연구에서 pinosylvin 및 유도체 3-hydroxy-5-(2-ethyl-pentanoyl)-trans-stilbene (HEPTS)가 prostaglandin E₂ (PGE₂) 및 nitric oxide 생성을 동시에 억제하는 것을 확인하였다. 따라서 pinosylvin 및 HEPTS의 염증을 통한 암화 진행의 억제 효능을 in vitro 및 in vivo 실험기법을 이용하여 확인하였다. 발암물질 7,12-dimethylbenz[a]anthracene (DMBA)과 염증 유발 및 발암 촉진제 12-O-Tetradecanoylphorbol 13-acetate (TPA)로 유도한 다단계 생쥐 피부암 모델에서 pinosylvin은 종양 (papilloma)의 발생률 및 종양 수의 증가를 효과적으로 억제하였다. 또한 HEPTS는 RAW 264.7 세포에서 TIR-containing adapter molecule (TRIF)-의존적 신호전달체계를 저해함으로써 inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2)의 mRNA, 단백질 발현을 조절하여 항염증 및 암예방제로서의 가능성을 제시하였다. 또한 in vivo DMBA/TPA 피부암 모델에서 pinosylvin과 마찬가지 효능을 보였다. 이러한 결과를 바탕으로 pinosylvin 및 HEPTS가 암화 단계, 특히 암진행 단계에서 염증과 관련된 인자들의 작용을 저해함으로써 암예방 활성을 나타낼 수 있을 것으로 전망된다. 다음, Pinosylvin 및 HEPTS의 대장직장암 세포에 대한 암세포 증식 저해 활성을 평가하고 그 작용기전을 연구하였다. 우선, pinosylvin은 G0/G1 세포 주기 정체를 유도하였으며 이는 G1기에 관여하는 c-Myc, cyclin 및 cyclin dependent kinase (CDK) 단백질의 발현 저해, p21, p53의 발현 증가, retinoblastoma protein (pRb)의 인산화 저해 등의 작용에 기인하는 것으로 여겨진다. 또한 세포 신호전달 물질 중 Akt, β-catenin 등의 활성화 저해로 인한 다양한 하위 단계 세포증식 및 종양관련 인자들의 발현이 저해되고 암억제 인자들의 발현이 증가되는 활성을 나타내었다. 이러한 다양한 작용을 통하여 pinosylvin은 암세포 성장저해 활성을 나타낸다고 할 수 있다. HEPTS의 경우 저농도에서는 G1 세포 주기 정체를 유도하고 고농도에서는 apoptosis를 유도하였다. Apoptosis는 Annexin/PI 이중염색을 통해 재차 확인하였으며, G1기에 관여하는 다양한 인자들의 발현 조절 및 Akt, β-catenin, STAT3와 같은 인자들의 저해 활성을 확인하였다. 또한 COX-2를 stable transfection한 HCT 116세포에서의 pinosylvin 및 HEPTS의 COX-2 발현 저해 활성을 확인할 수 있었다. 암세포 전이 촉진과 관련하여 matrix metalloproteinase (MMP) 류 및 tissue inhibitor of metalloproteinase (TIMP) 류에 대한 pinosylvin 및 DMS의 저해 활성을 확인한 후, migration과 관련된 실험을 통해 재차 저해 활성을 확인 한 후 생쥐 폐암 전이모델에서의 활성을 평가하였다. 그 결과 nodule 생성 및 폐 조직의 무게를 감소시켰다. 지금까지의 실험결과를 토대로 본 연구는, pinosylvin 및 그 유도체인 DMS, HEPTS는 암화의 각 단계에 다양하게 작용하는 새로운 암예방 효능 물질로서의 가능성을 제시한다고 여겨진다.