• Biomolecules & Therapeutics
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• 제30권6호
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• pp.553-561
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• 2022

Chronic myeloid leukemia (CML) is a slowly progressing hematopoietic cell disorder. Sphingosine kinase 1 (SPHK1) plays established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers, including leukemia. However, small-molecule inhibitors targeting SPHK1 in CML still need to be developed. This study revealed the role of SPHK1 in CML and investigated the potential anti-leukemic activity of hirsuteine (HST), an indole alkaloid obtained from the oriental plant Uncaria rhynchophylla, in CML cells. These results suggest that SPHK1 is highly expressed in CML cells and that overexpression of SPHK1 represents poor clinical outcomes in CML patients. HST exposure led to G2/M phase arrest, cellular apoptosis, and downregulation of Cyclin B1 and CDC2 and cleavage of Caspase 3 and PARP in CML cells. HST shifted sphingolipid rheostat from sphingosine 1-phosphate (S1P) towards the ceramide coupled with a marked inhibition of SPHK1. Mechanistically, HST significantly blocked SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways. In addition, HST can be docked with residues of SPHK1 and shifts the SPHK1 melting curve, indicating the potential protein-ligand interactions between SPHK1 and HST in both CML cells. SPHK1 overexpression impaired apoptosis and proliferation of CML cells induced by HST alone. These results suggest that HST, which may serve as a novel and specific SPHK1 inhibitor, exerts anti-leukemic activity by inhibiting the SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt pathways in CML cells, thus conferring HST as a promising anti-leukemic drug for CML therapy in the future.

• Journal of Ginseng Research
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• 제46권3호
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• pp.396-407
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• 2022

Background: Colorectal cancer (CRC) has a high morbidity and mortality worldwide. 20 (S)-ginsenoside Rh2 (G-Rh2) is a natural compound extracted from ginseng, which exhibits anticancer effects in many cancer types. In this study, we demonstrated the effect and underlying molecular mechanism of G-Rh2 in CRC cells in vitro and in vivo. Methods: Cell proliferation, migration, invasion, apoptosis, cell cycle, and western blot assays were performed to evaluate the effect of G-Rh2 on CRC cells. In vitro pull-down assay was used to verify the interaction between G-Rh2 and Axl. Transfection and infection experiments were used to explore the function of Axl in CRC cells. CRC xenograft models were used to further investigate the effect of Axl knockdown and G-Rh2 on tumor growth in vivo. Results: G-Rh2 significantly inhibited proliferation, migration, and invasion, and induced apoptosis and G₀/G₁ phase cell cycle arrest in CRC cell lines. G-Rh2 directly binds to Axl and inhibits the Axl signaling pathway in CRC cells. Knockdown of Axl suppressed the growth, migration and invasion ability of CRC cells in vitro and xenograft tumor growth in vivo, whereas overexpression of Axl promoted the growth, migration, and invasion ability of CRC cells. Moreover, G-Rh2 significantly suppressed CRC xenograft tumor growth by inhibiting Axl signaling with no obvious toxicity to nude mice. Conclusion: Our results indicate that G-Rh2 exerts anticancer activity in vitro and in vivo by suppressing the Axl signaling pathway. G-Rh2 is a promising candidate for CRC prevention and treatment.

• 한국식품과학회지
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• 제54권1호
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• pp.28-34
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• 2022

본 연구에서는 진피 소수성 추출물(CECU)의 U87MG 교모세포종 암줄기세포에 대한 항암 활성을 확인하였다. 그 결과, CECU는 25-200 ㎍/mL의 농도 범위에서 U87MG 교모세포종 암줄기세포의 증식, 종양구체 형성과 이동능력을 유의적으로 저해하였다. 특히, CECU는 G0/G1기에서 세포주기 정지와 세포사멸을 유도하여 교모세포종 암줄기세포의 증식을 억제할 수 있었다. 게다가, 교모세포종 암줄기세포에 대한 CECU의 항암 활성은 CD133, Oct4, Nanog, Integrin α6, ALDH1A1과 같은 줄기세포능 조절인자들의 발현과 STAT3 신호전달경로를 저해함으로써 유도된 것임을 확인하였다. 마지막으로, CAM assay를 통해 CECU가 U87MG 교모세포종 암줄기세포의 in vivo 종양 형성을 효과적으로 억제함을 입증하였다. 따라서, 본 연구는 진피 소수성 추출물이 주요 stemness marker들의 발현과 핵심 stemness 조절 신호전달경로를 억제함으로써 U87MG 교모세포종 암줄기세포에 대한 항암 활성을 나타냄을 입증하여, 교모세포종의 예방 및 치료를 위한 천연물 소재로서의 활용 가능성을 새롭게 제시하였다.

• Biomolecules & Therapeutics
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• 제32권1호
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• pp.123-135
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• 2024

Although gemcitabine-based regimens are widely used as an effective treatment for pancreatic cancer, acquired resistance to gemcitabine has become an increasingly common problem. Therefore, a novel therapeutic strategy to treat gemcitabine-resistant pancreatic cancer is urgently required. Piceamycin has been reported to exhibit antiproliferative activity against various cancer cells; however, its underlying molecular mechanism for anticancer activity in pancreatic cancer cells remains unexplored. Therefore, the present study evaluated the antiproliferation activity of piceamycin in a gemcitabine-resistant pancreatic cancer cell line and patient-derived pancreatic cancer organoids. Piceamycin effectively inhibited the proliferation and suppressed the expression of alpha-actinin-4, a gene that plays a pivotal role in tumorigenesis and metastasis of various cancers, in gemcitabine-resistant cells. Long-term exposure to piceamycin induced cell cycle arrest at the G₀/G₁ phase and caused apoptosis. Piceamycin also inhibited the invasion and migration of gemcitabine-resistant cells by modulating focal adhesion and epithelial-mesenchymal transition biomarkers. Moreover, the combination of piceamycin and gemcitabine exhibited a synergistic antiproliferative activity in gemcitabine-resistant cells. Piceamycin also effectively inhibited patient-derived pancreatic cancer organoid growth and induced apoptosis in the organoids. Taken together, these findings demonstrate that piceamycin may be an effective agent for overcoming gemcitabine resistance in pancreatic cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10407-10412
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• 2015

Background: ${\beta}$-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ${\beta}$-elemene against glioma cells remains unclear. In the present study, we assessed effects of ${\beta}$-elemene on human glioma cells and explored the underlying mechanism. Materials and Methods: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ${\beta}$-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ${\beta}$-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ${\beta}$-elemne treatment group. Results: With increase in the concentration of ${\beta}$-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability ($IC_{50}$) was $48.5{\mu}g/mL$ for 24h. ${\beta}$-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ${\beta}$-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ${\beta}$-elemene in a time and does-dependent manner. Conclusions: Our results indicate that ${\beta}$-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제14권7호
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• pp.4421-4426
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• 2013

Objective: The present study employed 5-aza-2'-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene. Methods: Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR, a specific demethylating agent, for 24, 48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression. Results: MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 ${\mu}mol/L$ 5-Aza-CdR). After treatment with 0, 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was $69.7{\pm}0.99%$, $76.1{\pm}0.83%$, $83.8{\pm}0.35%$, $95.5{\pm}0.55%$ respectively (P<0.05), and the proportion in S was $29.8{\pm}0.43%$, $23.7{\pm}0.96%$, $15.7{\pm}0.75%$, $1.73{\pm}0.45%$, respectively (P<0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 ${\mu}mol/L$ 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were $1{\pm}0$, $1.49{\pm}0.14$, $1.86{\pm}0.09$ and $5.80{\pm}0.15$ (P<0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were $0.12{\pm}0.01$, $0.23{\pm}0.02$, $0.31{\pm}0.02$, $0.62{\pm}0.03$ (P<0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration. Conclusions: TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.

• 한국동물위생학회지
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• 제37권2호
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• pp.111-122
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• 2014

본 연구는 대나무 분말의 항산화 활성이 비육돈에서 면역조절 효과에 미치는 영향을 구명하기 위해 실시하였으며, 이를 위해 대나무 분말 추출물의 항산화능을 검정하기 위하여, 총페놀 함량, 플라보노이드 함량 및 DHHP, ABST, hydroxyl radical 등의 소거활성능을 알아보았다. 대나무 추출물의 항산화능은 높게 나타났으며, DPPH와 ABTS, HRSA 라디칼 소거능은 12~21%였으며, 추출물의 유효성분으로 총페놀 함량은 171 mg/g이었으며, 플라보노이드 함량은 127. mg/g으로 나타났다. 다음으로 대나무 분말이 함유된 배합사료 급여시 돼지의 면역력에 미치는 영향을 알아보았다. 처리구에는 일반사료를 급여하였고, 시험구는 일반사료에 대나무분말을 1.0%, 2.0% 및 3.0%을 첨가하여 급여하였다. 대나무분말 첨가수준에 따라 혈액의 혈구계수 중 백혈구, 적혈구, 헤마토크리트 함량에서 유의적인 수준의 차이를 나타내지 않았다. 하지만 헤모글로빈 함량이 무처리 대조구에 비교하여 증가하였으며 특히 2~3%의 처리구에서 헤모글로빈 함량은 높게 나타났다. 혈액 내 총단백질, 혈중 알부민의 농도를 분석한 결과 처리구와 무처리구간에 유의적인 차이를 나타내지 않았다. AST, ALT 함량은 대나무 분말 처리군에서 무처리군보다 약간 높게 나타나지만 정상적인 범위내에서의 변화를 보였다. immunoglobulin에서 IgA와 IgG 농도는 무처리구에 비교하여 대나무 분말 처리구에서 높게 나타나고, IgA는 처리구 간의 차이가 나타나지 않았지만 IgG농도에서는 대나무 추출물 2%, 3% 처리구가 1% 처리구에 비해 높은 수준을 나타냈다. TNF-${\alpha}$의 경우 무첨가구와 첨가구 사이의 유의적인 차이가 없었지만, 대나무 분말 첨가구에서 interferon-${\gamma}$의 함량은 모든 첨가구에서 무처리 군보다 유의적으로 높게 나타났다. 특히 2%, 3% 처리구가 1%의 처리구 보다도 높은 interferon-${\gamma}$의 함량을 나타내었다. 이는 대나무의 페놀, 또는 플라보노이드 성분에 기초한 높은 항산화력에 의하거나, 또는 대나무에 포함된 돼지 초기 면역반응을 활성화시킬 수 있는 물질에 의한 면역활성 증강에 따른 것으로 사료된다. 결론적으로 대나무 분말의 높은 항산화력은 돼지 면역력 향상에 기여하였으며, 특히 2~3% 대나무 분말 첨가 사료는 돼지 생리활성을 크게 증강시키는 것으로 사료된다.