The autophagy inhibitor verteporfin moderately enhances the antitumor activity of gemcitabine in a pancreatic ductal adenocarcinoma model

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to chemotherapy. It has been described as requiring elevated autophagy for growth and inhibiting autophagy has been proposed as a treatment strategy. To date, all preclinical reports and clinical trials investigating pharmacological inhibition of autophagy have used chloroquine or hydroxychloroquine, which interfere with lysosomal function and block autophagy at a late stage. Verteporfin is a newly discovered autophagy inhibitor that blocks autophagy at an early stage by inhibiting autophagosome formation. Here we report that PDAC cell lines show variable sensitivity to verteporfin in vitro, suggesting cell-line specific autophagy dependence. Using image-based and molecular analyses, we show that verteporfin inhibits autophagy stimulated by gemcitabine, the current standard treatment for PDAC. Pharmacokinetic and efficacy studies in a BxPC-3 xenograft mouse model demonstrated that verteporfin accumulated in tumors at autophagy-inhibiting levels and inhibited autophagy in vivo, but did not reduce tumor volume or increase survival as a single agent. In combination with gemcitabine verteporfin moderately reduced tumor growth and enhanced survival compared to gemcitabine alone. While our results do not uphold the premise that autophagy inhibition might be widely effective against PDAC as a single-modality treatment, they do support autophagy inhibition as an approach to sensitize PDAC to gemcitabine.

Keywords: autophagy; chemosensitization; gemcitabine; pancreatic cancer; verteporfin.

Fig 1

Effect of long term verteporfin treatment on PDAC cell line viability in vitro.(A) The eight cell lines were treated with 0-10µM verteporfin (VP) for up to 7 days and cell proliferation was monitored at the indicated time points. Media and drugs were replenished every third day. Capan 1 and Capan 2 cell viability and proliferation was assayed using MTT and measuring OD 570. Other cell lines were stained with Hoechst 33342 and nuclei were quantified using a Cellomics ArrayScan VTI automated fluorescence microscope. (mean ± S.D. (error bars), n=4) (B) BxPC-3, SU86.86, and MIA PaCa-2 cells were exposed to vehicle control or 100nM bafilomycin A1 in complete medium for 4h. Lysates were collected and immunoblotted for LC3 or p62. β-tubulin was monitored as a loading control.

Fig 2

Inhibition of gemcitabine-induced autophagy by verteporfin. (A-C) MCF-7 EGFP-LC3 cells were exposed to 500nM gemcitabine (Gem) for 24h in the presence or absence of 10µM verteporfin, or to 0.1% DMSO vehicle control. Cells were fixed and stained with Hoechst 33342 and punctate EGFP-LC3 fluorescence was (A) visualized and (B) quantified using a Cellomics ArrayScan VTI automated fluorescence microscope. (* p < 0.01, Student's t-test)(mean ± S.D (error bars), n=3). (C) MCF-7 EGFP-LC3 cells were exposed to 0.1% DMSO, 500nM gemcitabine, or 30nM rapamycin, a known stimulator of autophagy, for 24h in the absence or presence of 10µM verteporfin. Lysates were collected and immunoblotted for EGFP. (D) BxPC-3 cells were exposed to 500nM gemcitabine for 24h in the presence or absence of 10µM verteporfin, 100nM bafilomycin A1, or 0.1% DMSO vehicle control. Lysates were collected and immunoblotted for LC3 and p62. β-tubulin was monitored as a loading control.

Fig 3

Effect of gemcitabine treatment on selected PDAC cell lines and tumour xenografts. (A) BxPC-3 and SU86.86 cells were treated with 50-1000nM gemcitabine (Gem) in vitro for up to 7 days. Media and drugs were replenished every third day. Cell viability and proliferation was measured as in Fig. 1A (mean ± S.D (error bars), n=4). (B) BxPC-3 and SU86.86 tumor-bearing mice were treated with saline as a control or with gemcitabine at 120mg/kg or 240mg/kg i.p. once per week over a period of four weeks. Treatment was initiated when tumors reached 100-150 mm³ (day 24 for BxPC-3 and day 25 for SU86.86). Tumor growth was monitored using digital callipers. Tumor growth is presented as relative tumor size, where tumor volumes were normalized with respect to the tumor volume of each mouse on the initial day of treatment and the average of each group was plotted. Arrows indicate the last day of treatment for each tumor model. (mean ± S.E.M (error bars), n=6 for all groups at the start of the study).

Fig 4

Verteporfin accumulation in vivo. BxPC-3 tumor-bearing Rag2M mice were treated with verteporfin at 45mg/kg i.p. (A) Tumors and (B) blood samples were harvested 2, 8, 16, and 24h after administration for pharmacokinetic analysis. (mean ± S.D. (error bars), n=3).

Fig 5

Effect of verteporfin on BxPC-3 tumour xenograft tissue. BxPC-3 tumors harvested 2, 8, 16, and 24h after a single verteporfin administration at 45mg/kg were also subjected to western blot analysis. Tumor sections were homogenized and immunoblotted for p62. In a separate experiment, the same lysates were probed for LC3. β-tubulin was monitored as a loading control for both experiments (n=2 per group).

Fig 6

Anticancer efficacy of verteporfin, gemcitabine and the combination in in a BxPC-3 tumor model. BxPC-3 tumor-bearing Rag2M mice were given i.p. administrations of (A) a combination of 45mg/kg verteporfin and 120mg/kg gemcitabine or each drug on its own or (B) a combination of 45mg/kg verteporfin and 240mg/kg gemcitabine or each drug on its own for a period of four weeks. Treatment was initiated when tumors reached 100-150mm³, day 25. DSPE-PEG micelles without drug were delivered as the control. Tumor growth is presented as both average tumor volume (top) and relative tumor size (middle) as in Fig. 3B. Kaplan-Meier survival curves (bottom) illustrate the number of surviving mice in treatment group post drug administration. Arrows indicate the last day of treatment. (** p < 0.01)(mean ± S.E.M (error bars), n=8 for all groups at the start of the study).