Kangwook Lee

SH003 represses tumor angiogenesis by blocking VEGF binding to VEGFR2

Tumor angiogenesis is a key feature of cancer progression, because a tumor requires abundant oxygen and nutrition to grow. Here, we demonstrate that SH003, a mixed herbal extract containing Astragalus membranaceus (Am), Angelica gigas (Ag) and Trichosanthes Kirilowii Maximowicz (Tk), represses VEGF-induced tumor angiogenesis both in vitro and in vivo. SH003 inhibited VEGF-induced migration, invasion and tube formation in human umbilical vein endothelial cells (HUVEC) with no effect on the proliferation. SH003 reduced CD31-positive vessel numbers in tumor tissues and retarded tumor growth in our xenograft mouse tumor model, while SH003 did not affect pancreatic tumor cell viability. Consistently, SH003 inhibited VEGF-stimulated vascular permeability in ears and back skins. Moreover, SH003 inhibited VEGF-induced VEGFR2-dependent signaling by blocking VEGF binding to VEGFR2. Therefore, our data conclude that SH003 represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation, and suggest that SH003 may be useful for treating cancer.

DSGOST inhibits tumor growth by blocking VEGF/VEGFR2-activated angiogenesis

Tumor growth requires a process called angiogenesis, a new blood vessel formation from pre-existing vessels, as newly formed vessels provide tumor cells with oxygen and nutrition. Danggui-Sayuk-Ga-Osuyu-Saenggang-Tang (DSGOST), one of traditional Chinese medicines, has been widely used in treatment of vessel diseases including Raynauds syndrome in Northeast Asian countries including China, Japan and Korea. Therefore, we hypothesized that DSGOST might inhibit tumor growth by targeting newly formed vessels on the basis of its historical prescription. Here, we demonstrate that DSGOST inhibits tumor growth by inhibiting VEGF-induced angiogenesis. DSGOST inhibited VEGF-induced angiogenic abilities of endothelial cells in vitro and in vivo, which resulted from its inhibition of VEGF/VEGFR2 interaction. Furthermore, DSGOST attenuated pancreatic tumor growth in vivo by reducing angiogenic vessel numbers, while not affecting pancreatic tumor cell viability. Thus, our data conclude that DSGOST inhibits VEGF-induced tumor angiogenesis, suggesting a new indication for DSGOST in treatment of cancer.

SH003 suppresses breast cancer growth by accumulating p62 in autolysosomes

Drug markets revisits herbal medicines, as historical usages address their therapeutic efficacies with less adverse effects. Moreover, herbal medicines save both cost and time in development. SH003, a modified version of traditional herbal medicine extracted from Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes Kirilowii Maximowicz (Tk) with 1:1:1 ratio (w/w) has been revealed to inhibit tumor growth and metastasis on highly metastatic breast cancer cells, both in vivo and in vitro with no toxicity. Meanwhile, autophagy is imperative for maintenance cellular homeostasis, thereby playing critical roles in cancer progression. Inhibition of autophagy by pharmacological agents induces apoptotic cell death in cancer cells, resulting in cancer treatment. In this study, we demonstrate that SH003-induced autophagy via inhibiting STAT3 and mTOR results in an induction of lysosomal p62/SQSTM1 accumulation-mediated reactive oxygen species (ROS) generation and attenuates tumor growth. SH003 induced autophagosome and autolysosome formation by inhibiting activation of STAT3- and mTOR-mediated signaling pathways. However, SH003 blocked autophagy-mediated p62/SQSTM1 degradation through reducing of lysosomal proteases, Cathepsins, resulting in accumulation of p62/SQSTM1 in the lysosome. The accumulation of p62/SQSTM1 caused the increase of ROS, which resulted in the induction of apoptotic cell death. Therefore, we conclude that SH003 suppresses breast cancer growth by inducing autophagy. In addition, SH003-induced p62/SQSTM1 could function as an important mediator for ROS generation-dependent cell death suggesting that SH003 may be useful for treating breast cancer.

Inhibitory effect of ucha-shinki-hwan on cold-mediated response in human dermal microvascular endothelial cells

Raynauds phenomenon is characterized by prolonged vasoconstriction in cutaneous capillaries on cold stress. RhoA activity would be a therapeutic target for treating Raynauds phenomenon. A traditional herbal medicine, ucha‐shinki‐hwan, has been used to promote vasodilation, but the biological mechanism of ucha‐shinki‐hwan is still unclear. Thus, we hypothesized that ucha‐shinki‐hwan is able to be used for treatment of Raynauds phenomenon and that ucha‐shinki‐hwan inhibits cold‐induced vasoconstriction by targeting RhoA GTPase.

Abstract 4010: Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in tumor microenvironment

The critical roles of inflammation in the development of cancer have long been appreciated. A growing body of evidence supports the notion that infiltrates of inflammatory cells into tumor microenvironment influence the tumor progression by providing bioactive molecules including pro-inflammatory cytokines. Importantly, the increased number of mast cells within tumor microenvironment has been associated with a poor survival in cancer patients. Moreover, keratinocyte inflammation is known to be crucial for skin tumor development. The use of natural products to reduce inflammation in tumor microenvironment is gaining an interest, because of their reduced toxicity toward normal cells. In this study, we tested the effects of Tonggyu-tang (TGT) which is composed of 14 different herbal extracts on the activity of mast cells. We found that TGT significantly reduced the expression and production of inflammatory cytokines such as IL-4, IL-6, IL-8, and TNF-α in PMA and ionomycin- stimulated HMC-1 (human mast cell line-1). In an attempt to determine molecular mechanism underlying the inhibitory effects of TGT on cytokine expression, we revealed that TGT suppressed MAPK signaling pathway including ERK, p38, and JNK as well as NF-κB pathway, which are known to regulate inflammatory cytokine expression. Similar results were obtained from the LPS-stimulated HaCaT cells, immortalized human keratinocytes. Taken together, our results demonstrate that TGT suppresses inflammation by inhibiting the expression of pro-inflammatory cytokine in both mast cells and keratinocytes, thereby potentially leading to inhibition of tumor progression. Citation Format: Hyoin Kim, Seong-Gyu Ko, Yong Cheol Shin, Ji Hye Kim, Hye-Sook Seo, Tai Young Kim, Se Hyang Hong, Kangwook Lee, Jin Mo Ku, Myeong-Sun Kim, Yu-Jeong Choi, Soo-yeon Kang, Chunhoo Cheon, Youme Ko, Huang Ching Wen, Yui Sasaki, Sohyeon Kang. Tonggyu-tang, a traditional Korean medicine, suppresses inflammation, potential implications in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4010. doi:10.1158/1538-7445.AM2017-4010

Abstract 4310: SH003 induces apoptosis of DU145 prostate cancer cells by inhibiting ERK-involved pathway

Herbal medicines have been used in cancer treatment, with many exhibiting favorable side effect and toxicity profiles compared with conventional chemotherapeutic agents. SH003 is a novel extract from Astragalus membranaceus, Angelica gigas, and Trichosanthes Kirilowii Maximowicz combined at a 1:1:1 ratio that impairs the growth of breast cancer cells. Our data demonstrate that SH003 induced apoptosis in DU145 prostate cancer cells by inhibiting ERK signaling. SH003 induced apoptosis of prostate cancer cells in dose-dependent manner, which was independent of androgen dependency. SH003 also increased intracellular ROS levels but this is not associated with its pro-apoptotic effects. SH003 inhibited phosphorylation of Ras/Raf1/MEK/ERK/p90RSK in androgen-independent DU145 cells, but not androgen-dependent LNCaP and PC-3 cells. Moreover, ERK2 overexpression rescued SH003-induced apoptosis in DU145 cells. Thus, our data conclude that SH003 induces apoptotic cell death of DU145 prostate cancer cells by inhibiting ERK-mediated pathway. Citation Format: Yu-Jeong Choi, Myeong-Sun Kim, Soo-Yeon Kang, Kangwook Lee, Jin Mo Ku, Se Hyang Hong, Hyo In Kim, Chunhoo Cheon, Youme Ko, Huang Ching Wen, Yui Sasaki, Sohyeon Kang, Tai Young Kim, Ji Hye Kim, Yong Cheol Shin, Seong-Gyu Ko. SH003 induces apoptosis of DU145 prostate cancer cells by inhibiting ERK-involved pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4310. doi:10.1158/1538-7445.AM2017-4310

Abstract 3522: Cyclins and CDKs regulation and caspase cascade activation by cucurbitacin D induced cell cycle arrest and apoptosis in pancreatic tumor

Pancreatic cancer has a poor prognosis and very low survival rate over the world. Because pancreatic cancer probably is diagnosed at a late stage, aggressive local invasion, and poor response to chemotherapy. Gemcitabine was the standard treatment for advanced and metastatic pancreatic cancer patients, but it is associated with multiple adverse effects-fever, fatigue, nausea, and drug resistance. Whether cucurbitacin D has any efficacy against human pancreatic cancer was examined in cell culture system. In vitro, cell viability was measured by MTT assay to recognize of cell cytotoxicity. Consequently, cytotoxicity was observed at a low concentration of cucurbitacin D. Wound healing assay and clonogenic assay indicated that cucurubitacin D inhibited the growth of cell growth through cyclins and CDKs regulation, and decreased colony-forming ability. Also, this compound down-regulated expression level of anti-apoptotic protein, Bcl-2, up-regulated of pro-apoptotic molecule Bax, and activated caspase-8, caspase-3 cascade extrinsic pathway. Additionally, PARP, caspase-3 substrate, protein was cleaved by cucurbitacin D treatment. Overall, our study suggest that cucurbitacin D could be a clinical medicine for the treatment of pancreatic cancers. Citation Format: Myeong-Sun Kim, Ji Hye Kim, Jin Mo Ku, Se Hyang Hong, Kangwook Lee, Hyeong Sim Choi, Sang Mi Woo, Jee Yun Chang, Tai Young Kim, Seong Gyu Ko Ko. Cyclins and CDKs regulation and caspase cascade activation by cucurbitacin D induced cell cycle arrest and apoptosis in pancreatic tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3522.

Abstract 4543: Substrate trapping proteomics reveals novel mechanism for regulation of mTORC1 signaling by βTrCP-FNIP1/2-FLCN axis

Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2, a substrate adaptor for the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between βTrCP2 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCF (βTrCP2) E3 complex. Comparative mass spectrometry analysis of immunopurified βTrCP2 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. Interestingly, many novel substrates for βTrCP2, including TBC1D4, HCFC1, DENND4C, FNIP1, and FLCN are related to cell metabolism. TBC1D4 encodes a GTPase activating protein for the small GTPase Rab that controls insulin-dependent trafficking of the GLUT4 glucose transporter in adipocytes. DENND4C acts as a guanine nucleotide exchange factor for Rab10 and its activity is required for insulin-stimulated GLUT4 translocation to plasma membrane in adipocytes. HCFC1 is a member of the host cell factor family, affecting gluconeogenesis by modulating PGC-1α stability. These suggest βTrCP2 might play important roles in glucose homeostasis by regulating stability of several different target proteins. Here, in focused study, we found that βTrCP1/2 bound, polyubiquitylated, and destabilized FNIP1, FLCN interacting protein 1. We further demonstrated that FNIP1 degradation was promoted by AMPK activation after glucose depletion and expression of a degradation-resistant FNIP1 mutant results in sustained activation of mTORC1 signaling. Hence, our findings reveal that βTrCP1/2 is involved in nutrient sensing through the AMPK-FLCN-FNIP1 and mTORC1 signaling pathways. Citation Format: Tai Young Kim, Jee Yun Chang, Jin Mo Ku, Se Hyang Hong, ji Hye Kim, Hyeong Sim Choi, Kangwook Lee, Myeong-Sun Kim, Sang Mi Woo, Michael B. Major, Seong-Gyu Ko. Substrate trapping proteomics reveals novel mechanism for regulation of mTORC1 signaling by βTrCP-FNIP1/2-FLCN axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4543.