Cathepsin L inhibition by the small molecule KGP94 suppresses tumor microenvironment enhanced metastasis associated cell functions of prostate and breast cancer cells - PubMed (original) (raw)

Cathepsin L inhibition by the small molecule KGP94 suppresses tumor microenvironment enhanced metastasis associated cell functions of prostate and breast cancer cells

Dhivya R Sudhan et al. Clin Exp Metastasis. 2013 Oct.

Abstract

Metastasis remains the major cause of therapeutic failure, poor prognosis and high mortality in breast and prostate cancer patients. Aberrant microenvironments including hypoxia and acidic pH are common features of most solid tumors that have been long associated with enhanced metastasis and poor patient outcomes. Novel approaches to reduce metastatic incidences and improve overall survival of cancer patients clearly are needed. The crucial role of Cathepsin L (CTSL) in the dissemination of tumor cells has led to the development of novel cathepsin L inhibition strategies. The present study evaluated the ability of KGP94, a small molecule inhibitor of CTSL, to impair the metastatic phenotype of prostate (PC-3ML) and breast (MDA-MB-231) cancer cells both under normal and aberrant microenvironmental conditions. To assess the role of CTSL in hypoxia and acidosis triggered metastasis associated cell functions, secreted CTSL levels were determined under conditions pertinent to the tumor microenvironment. Acute exposures to hypoxic or acidic conditions significantly elevated secreted CTSL levels either through an increase in intracellular CTSL levels or through activation of lysosomal exocytosis or both, depending on the tumor type. Increases in CTSL secretion closely paralleled enhanced tumor cell migration and invasion suggesting that CTSL could be an essential factor in tumor microenvironment triggered metastasis. Importantly, KGP94 treatment led to marked attenuation of tumor cell invasion and migration under both normal and aberrant microenvironmental conditions suggesting that it may have significant utility as an anti-metastatic agent.

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Conflict of interest statement

Disclosure of potential conflicts of interest: The authors have no conflict of interest to disclose.

Figures

Fig. 1. Cathepsin L secretion and activity positively correlates with invasive potential

A and B Intracellular CTSL levels in prostate and breast cancer cells were determined by performing Western blots using whole cell lysates. C and D, CTSL secretion levels of prostate and breast cancer cells were determined by performing ELISA on cell conditioned media. Means and standard errors of three independent experiments are shown. E and F, Ratios of secreted CTSL to cystatin C levels for prostate and breast cancer cells were determined by performing ELISA on cell conditioned media. Ratios shown are relative levels normalized to least invasive prostate and breast cancer cell lines, respectively.

Fig. 2. KGP94 abrogates CTSL activity and metastatic phenotype of prostate and breast cancer cells

A, The effect of 24 h treatment with KGP94 on the clonogenicity of PC-3 cells was evaluated 2 weeks later. (*) p<0.01. B The effect of KGP94 treatment on the activity of CTSL secreted byPC-3ML and MDA-MB-231 cells was assessed by incubating cell conditioned media with a fluorogenic CTSL substrate Z-Phe-Arg-AMC in the absence (black bars) or presence (grey bars) of 25 µM KGP94. Activity is expressed as relative fluorescence units and results of three independent experiments are shown. (***) p<0.0001. C and D, PC-3, MDA-MB-231 and CTSL knock down PC-3 cells were seeded into transwell migration chambers in the presence or absence of KGP94 and the number of migrated cells was enumerated 24 h later. Mean and standard error values of three independent experiments are shown. (*) p<0.05, (***) p<0.0005. E and F, The impact of KGP treatment on PC-3ML and MDA-MB-231 cell transwell invasion is shown. Results are mean and standard error values of three independent experiments. (**) p<0.01, (***) p< 0.001.

Fig. 3. Acute exposure to hypoxia enhances CTSL secretion

A and B, CTSL secreted levels in PC-3ML and MDA-MB-231 cells exposed to hypoxic conditions (1% O2) for the indicated durations followed by reoxygenation for a total time of 24 h. Secreted CTSL levels were determined by ELISA on cell conditioned media and normalized to cell numbers. Shown are mean and standard error values calculated from three independent experiments. (*) p<0.05, (**) p<0.005, (***) p<0.001 C and D, Western blot analysis of PC-3ML and MDA-MB-231 cells exposed to hypoxia for various durations. E, Lysosomal localization in response to exposure to hypoxia was determined by immunostaining for lysosomal marker protein LAMP-1. F, The effect of hypoxia and reoxygenation on exocytosis of lysosomal content was determined by quantifying the lysosomal marker enzyme β-Hexosaminidase activity in the media. Results from three independent experiments are shown. (*) p<0.05, (**) p<0.01.

Fig. 4. Acute exposure to an acidic extracellular environment enhances CTSL secretion

A and B, CTSL secretion levels in PC-3ML and MDA-MB-231 cells pre-exposed to acidic conditions for the indicated durations followed by incubation under neutral pH conditions for 24 h. Secreted CTSL levels were determined by performing ELISA on cell conditioned media and normalized to cell numbers. Results from three independent experiments are shown. (*) p<0.05, (**) p<0.01. C and D, Western blot analysis of PC-3ML and MDA-MB-231 cells exposed to 6.8pH for various durations. E, Effect of acidic exposure on lysosomal localization in response to acidic extracellular condition was determined by immunostaining for lysosomal marker protein LAMP-1. F, Exocytosis of lysosomal content was determined by quantifying of β- Hexosaminidase activity in conditioned media. Results are mean and standard errors from three independent experiments. (*) p<0.05, (**) p<0.01.

Fig. 5. Acute hypoxia and acidosis augments metastatic phenotype

A, PC-3ML and MDA-MB-231 cells were exposed to 1% O2 for indicated durations and then allowed to migrated under normoxic conditions. The number of migrated cells was quantified 24 h later. Mean and standard error from three independent experiments are shown. (***) p<0.0001. B, PC-3ML monolayers were exposed to hypoxic conditions following which a scratch was made on the monolayers. Cells were incubated under normoxic conditions and imaged at 5× magnification 24 h later. C and D, PC-3ML and MDA-MB-231 cells were seeded into invasion inserts and exposed to hypoxia for indicated durations. 24 h later, invaded cells were stained and counted. Mean and standard error are shown. (*) p<0.05, (**) p<0.005, (***) p<0.001. E, PC-3ML and MDA-MB-231 cells were briefly exposed to 6.8pH, maintained under neutral conditions for 24 h and the number of migrated cells was quantified. Mean and standard errors for three independent experiments are shown. (**) p<0.005, (***), p<0.0001. F, Wound healing in response to acidic extracellular condition was assessed as mentioned in 5B. G and H, PC-3ML and MDA-MB-231 cells were exposed to pH 6.8 for indicated durations and seeded into invasion inserts. 24 h later, invaded cells were stained and counted. Mean and standard error are shown. (*) p<0.05, (***) p<0.001.

Fig. 6. KGP94 obliterates acidosis and hypoxia triggered invasiveness

A and B, PC-3ML and MDA-MB-231 cells were exposed to hypoxic conditions for 4 h. Cells were seeded into invasion inserts and allowed to invade for 24 h in the presence of indicated doses of KGP94. Results from three independent experiments are shown. (***) p<0.0001. C and D, PC-3ML and MDA-MB-231 cells were exposed to pH 6.8 for 4 h, seeded into invasion inserts and allowed to invade for 24 h in the presence of indicated doses of KGP94. (*) p<0.05, (**) p< 0.01.

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Cathepsin L inhibition by the small molecule KGP94 suppresses tumor microenvironment enhanced metastasis associated cell functions of prostate and breast cancer cells - PubMed (original) (raw)