High expression levels of unc-51-like kinase 1 as a predictor of poor prognosis in colorectal cancer

Zou, Yifeng; He, Xiaowen; He, Xiaosheng; Wu, Xianrui; Chen, Yufeng; Wu, Xiaojian; Wang, Jianping; Lan, Ping

doi:10.3892/ol.2015.3417

Cited by 26 publications

(19 citation statements)

References 30 publications

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“…In breast cancer [40] and gastric cancer [43], high expression of ULK-1 appears to associate to good patient prognosis. This contradicts the findings in colorectal cancer [44]. A separate study in colorectal cancer did not demonstrate any correlation to survivability, even after taking into account the status of KRAS.…”

Section: Ulk-1/ulk-2contrasting

confidence: 72%

Cell-Based Assays for Evaluation of Autophagy in Cancers

Pang¹,

Awi²,

Yap³

et al. 2019

Cell Culture

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Autophagy is a cellular mechanism that degrades damaged organelles and misfolded proteins to maintain cellular homeostasis. Autophagy in cancers is drawing increasing attentions due to its multifaceted roles in cancer development, progression, and treatment. There are several key autophagy effectors that are being extensively studied to understand the role of autophagy in cancer as well as their potential value as predictive and/or prognostic biomarkers and therapeutic target. These include ATG4A, ATG4B, Beclin-I, p62, LC3A, LC3B, LC3C, and LAMP. While having its own sophisticated pathway, autophagy has been reported to associate with multiple oncogenic pathways such as NF-kB, mTOR, and PI3K signaling. This chapter aims to provide a detailed protocol for researchers to investigate the role of autophagy using in vitro cell line as model. Here, we demonstrate several techniques including Western blot (WB), immunofluorescence (IF), and small-interfering RNA (siRNA) knockdown using colorectal cancer cell lines as samples. This chapter provides information to researchers especially those in their earlyand mid-career to plan and design their experiments to study the autophagy events in their area of interests. 1.In a 24-well plate, the cells were seeded at 50% confluency and left to incubate overnight.2. The media were then changed accordingly, with normal media, serum-free media, and media with the inhibitor. 3.After 24-hour incubation, the cells were collected and rinsed with PBS. 4.The cells were then lysed with lysis cocktail consisting of 1X RIPA lysis buffer and 1X protease inhibitor cocktail. 5.The lysates were vortexed briefly every 5 minutes for 30 minutes and kept in ice in between mixing. 6.The lysates were then centrifuged at 16000×g for 15 minutes at 4°C, and the supernatants were kept. 7.The lysates were then quantified by using BCA protein assay and immediately diluted with deionized water to obtain 13 μL of 20 μg lysate and then added to 5 μL of 4X SDS loading buffer and 2 μL of 2-mercaptoethanol.8. The samples were boiled at 90°C for 10 minutes, and 10 μL of the samples were loaded into the well of a gradient gel.9. The lysates were separated by SDS electrophoresis by using Tris/Glycine/SDS running buffer at 150 V for approximately 45 minutes and transferred to a PVDF membrane by using FlashBlot transfer buffer at 55 V for 1 hour. 10.The membrane was then blocked by using 5% Blotting Grade Blocker in TBST for 1 hour to prevent non-specific binding of antibody. 11.The membrane was then incubated with primary antibody diluted with the Blocker in TBST at 1:2000 dilution. 12.The incubation was done overnight at 4°C or 1 hour at room temperature.13. Any unbound antibodies were then removed by washing with TBST for 10 minutes once and 5 minutes for another 2 times. 14.Secondary antibodies conjugated with horseradish peroxidase (HRP) were then prepared by diluting the antibody in the Blocker in TBST at 1:3000 dilution. 15.The membrane was then incubated in the secondary antibody:a. Anti-rabbit secondary antib...

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Section: Ulk-1/ulk-2contrasting

confidence: 72%

Cell-Based Assays for Evaluation of Autophagy in Cancers

Pang¹,

Awi²,

Yap³

et al. 2019

Cell Culture

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“…Ulk1 is overexpressed in NSCLC cell lines. It has been reported that in several cancers, such as breast cancer, colorectal cancer, esophageal squamous cell carcinoma and human nasopharyngeal carcinoma, Ulk1 is upregulated and is negatively correlated with prognosis, but the role of Ulk1 in NSCLC remains unclear (12)(13)(14)(15)(16)). In the current study, the expression of Ulk1 was tested in five NSCLC cell lines (A549, H292, H460, H1299 and HCC827) and one normal human lung cell line (BEAS-2B).…”

Section: Resultsmentioning

confidence: 99%

“…Pike et al showed that Ulk1 promotes cell survival of several cancers (12). Moreover, overexpression of Ulk1 has been shown to be negatively correlated with the prognosis of a variety of cancers, such as colorectal cancer, breast cancer, human nasopharyngeal carcinoma and esophageal squamous cell carcinoma (12)(13)(14)(15)(16). Egan et al reported that the compound SBI0206965, a highly selective kinase inhibitor of Ulk1 both in vitro and in vivo, suppresses Ulk1-mediated phosphorylation events in cells and prevents Ulk1-dependent cell survival by suppressing autophagy (5).…”

Section: Introductionmentioning

confidence: 99%

SBI0206965, a novel inhibitor of Ulk1, suppresses non-small cell lung cancer cell growth by modulating both autophagy and apoptosis pathways

Tang

Yang

et al. 2017

Oncology Reports

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Lung cancer is a major public health problem worldwide. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases. Autophagy has recently sparked great interest, and it is thought to participate in a variety of diseases, including lung cancer. Uncoordinated (Unc) 51-like kinase 1 (Ulk1), a serine/threonine kinase, plays a central role in the autophagy pathway. However, the role of Ulk1 in NSCLC remains unclear. We report that NSCLC cell lines exhibited high expression of Ulk1 and that Ulk1 was negatively correlated with prognosis in lung cancer patients. Knockdown of Ulk1 or the inhibition of Ulk1 by the selective inhibitor SBI0206965, inhibited cell proliferation, induced cell apoptosis and enhanced the sensitivity of cisplatin against NSCLC cells. Moreover, we demonstrated that Ulk1 exerted oncogenic activity in NSCLC by modulating both autophagy and apoptosis pathways. Inhibition of autophagy by SBI0206965 sensitized NSCLC cells to cisplatin by inhibiting cisplatin induced cell-protective autophagy to promote apoptosis. Furthermore, SBI0206965 promoted apoptosis in NSCLC cells independent of autophagy, which was partly mediated by destabilization of Bcl2/Bclxl. In summary, our results show that inhibition of Ulk1 suppresses NSCLC cell growth and sensitizes NSCLC cells to cisplatin by modulating both autophagy and apoptosis pathways, and that Ulk1 might be a promising target for NSCLC treatment.

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“…Clinically, overexpression of ULK1 plays an oncogenic role in most of tumor types, including hepatocellular carcinoma [208], esophageal squamous cell carcinoma [209], nasopharygeal carcinoma [210] as well as colorectal cancer [211], which predicts poorer prognosis and severer progression. In addition, the protective autophagy induced by ULK1 is observed among mice with xenograft prostate cancer, thus preventing apoptosis and triggering neoplastic advancement [212].…”

Section: Functions Of Ampk Signaling Components In Tumorigenesismentioning

confidence: 99%

Functional characterization of AMP-activated protein kinase signaling in tumorigenesis

Cheng

Zhang

et al. 2016

Biochimica et Biophysica Acta (BBA) - Reviews on Cancer

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AMP-activated protein kinase (AMPK) is a ubiquitously expressed metabolic sensor among various species. Specifically, cellular AMPK is phosphorylated and activated under certain stressful conditions, such as energy deprivation, in turn to activate diversified downstream substrates to modulate the adaptive changes and maintain metabolic homeostasis. Recently, emerging evidences have implicated the potential roles of AMPK signaling in tumor initiation and progression. Nevertheless, a comprehensive description on such topic is still in scarcity, especially in combination of its biochemical features with mouse modeling results to elucidate the physiological role of AMPK signaling in tumorigenesis. Hence, we performed this thorough review by summarizing the tumorigenic role of each component along the AMPK signaling, comprising of both its upstream and downstream effectors. Moreover, their functional interplay with the AMPK heterotrimer and exclusive efficacies in carcinogenesis were chiefly explained among genetically altered mice models. Importantly, the pharmaceutical investigations of AMPK relevant medications have also been highlighted. In summary, in this review, we not only elucidate the potential functions of AMPK signaling pathway in governing tumorigenesis, but also potentiate the future targeted strategy aiming for better treatment of aberrant metabolism-associated diseases, including cancer.

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High expression levels of unc-51-like kinase 1 as a predictor of poor prognosis in colorectal cancer

Cited by 26 publications

References 30 publications

Cell-Based Assays for Evaluation of Autophagy in Cancers

Cell-Based Assays for Evaluation of Autophagy in Cancers

SBI0206965, a novel inhibitor of Ulk1, suppresses non-small cell lung cancer cell growth by modulating both autophagy and apoptosis pathways

Functional characterization of AMP-activated protein kinase signaling in tumorigenesis

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