Elsevier

Phytomedicine

Volume 27, 15 April 2017, Pages 1-7
Phytomedicine

Original article
Sulforaphane as an adjunctive to everolimus counteracts everolimus resistance in renal cancer cell lines

https://doi.org/10.1016/j.phymed.2017.01.016Get rights and content

Abstract

Background

The mechanistic target of rapamycin (mTOR) inhibitors, everolimus and temsirolimus, have widened therapeutic options to treat renal cell carcinoma (RCC). However, chronic treatment with these inhibitors often induces resistance, leading to therapeutic failure.

Purpose

The natural compound, sulforaphane (SFN), was added to an everolimus based regime in vitro in the hopes of preventing resistance development.

Methods

A panel of RCC cell lines (A498, Caki-1, KTCTL-26) was treated with everolimus or SFN or with an everolimus-SFN-combination, either short- (24 h) or long-term (8 weeks), and cell growth, proliferation, apoptosis, and cell cycle phases were measured. The cell cycle regulating proteins cdk1, cdk2, cyclin A, cyclin B, akt and raptor (both total and activated) were also evaluated.

Results

Short-term incubation with everolimus (1 nM) or SFN (5 µM) significantly reduced RCC cell growth. Additive effects on tumor growth and proliferation were evoked by the SFN-everolimus combination. Long-term everolimus-incubation led to resistance development in Caki-1 cells, evidenced by elevated growth and proliferation, associated with an increased percentage of G2/M (non-synchronized cell model) or S-phase (synchronized cell model) cells. Molecular analysis revealed up-regulation of the cdk1-cyclin B and cdk2-cyclin A axis, along with elevated phosphorylation of the mTOR sub-member, raptor. In contrast, resistance development was not observed with the long-term combination of SFN-everolimus. The combination suppressed Caki-1 growth and proliferation, and was associated with an increase in G0/G1-phase cells, diminished cdk1 and akt (both total and activated), cyclin B and raptor expression.

Conclusion

Adding SFN to an everolimus based RCC treatment regimen in vitro delayed resistance development observed with chronic everolimus monotherapy. Ongoing in vivo studies are necessary to verify the in vitro data.

Introduction

Increased knowledge about the molecular mechanisms involved in renal cell carcinoma (RCC) pathogenesis has led to the development and approval of a new class of therapeutics called targeted drugs. These targeted drugs, particularly the tyrosine kinase inhibitors sunitinib and sorafenib and the mechanistic target of rapamycin (mTOR) inhibitors temsirolimus and everolimus, have considerably widened the therapeutic spectrum and significantly improved progression-free survival. Still, they are not curative. Rather, all these agents almost inevitably fail, largely due to acquired resistance. In view of this obstacle, natural compounds have come into the focus of interest as potential complementary adjunctives to support “traditional” tumor therapy. In fact, use of complementary medicine (CM) together with conventional treatment is common among tumor patients. Between 40% (Üstündağ and Demir Zencirci, 2015) and 80% (Huebner et al., 2014, Saghatchian et al., 2014) of cancer patients have indicated that they apply CM in addition to conventional therapy. The most common reasons given for use of CM are dissatisfaction with conventional treatment, reduction of chemotherapeutic side effects (Gillett et al., 2012, Citrin et al., 2012), and to actively contribute to tumor therapy (Smith et al., 2016).

Despite the widespread use of CM, knowledge about its therapeutic potential is limited and only sparse information is available dealing with whether CM possesses potential in combating resistance development under chronic drug exposure. Therefore, an in vitro RCC cell culture protocol was designed to evaluate whether combined use of the phytochemical, sulforaphane (SFN), derived from cruciferous vegetables such as broccoli (Brassica oleracea), and the mTOR inhibitor everolimus might be superior to an everolimus monotherapy with respect to tumor growth and proliferation. SFN was chosen, since it has been demonstrated to interfere with cellular pathways involved in growth, apoptosis and motility, and suppresses tumor proliferation and metastasis in vitro and in vivo. High SFN intake has also been linked to a reduced cancer risk in several epidemiologic studies (Herr and Büchler, 2010, Higdon et al., 2007). A clinical trial has provided evidence that SFN down-regulates the prostate-specific antigen (PSA) level in men with prostate cancer, decreasing biochemical recurrence after radical prostatectomy (Cipolla et al., 2015). The relevance of SFN in RCC treated with an mTOR-inhibitor based regimen has to date not been evaluated. The current work points to SFN as a potent phytopharmaceutical, which might be of particular help in managing RCC treatment failure caused by chronic everolimus treatment.

Section snippets

Cell culture

Kidney carcinoma Caki-1 and KTCTL-26 cells were purchased from LGC Promochem, Wesel, Germany (Fogh et al., 1977, Högemann et al., 1994). A498 cells were derived from Cell Lines Service (Heidelberg, Germany). Caki-1 and KTCTL-26 cells were chosen, since both lines are derived from a clear cell renal cell carcinoma, which is the most common renal carcinoma tumor type. Both lines are VHL positive, whereas VHL function is disrupted in A498 cells. Tumor cells were grown and subcultured in RPMI 1640

Short-term combined SFN and everolimus additively reduces RCC growth and proliferation

5 nM everolimus, applied short-term for 48 h, induced a significant reduction in the number of Caki-1, KTCTL-26, and A498 cells (Fig. 1). A significant growth blocking effect was also observed when 5 µM SFN was applied, although this was less pronounced than that induced by 5 nM everolimus. Combined SFN-everolimus did not block cell growth better than did the everolimus monotherapy. Since 5 nM everolimus evoked a maximum effect, which could not be surpassed by adding SFN, the everolimus dosage was

Discussion

Despite improvements in treating RCC, resistance toward established drug regimens limits therapeutic efficacy of anti-tumor protocols. The results presented here, as well as results from other investigations, show that the mTOR inhibitor, everolimus, creates feedback loops during chronic treatment, leading to drug non-responsiveness (Juengel et al., 2014, Tsaur et al., 2012). Molecular analysis during non-responsiveness points to an everolimus driven enhanced expression of cdk1 and 2, cyclin A

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Acknowledgment

This work was supported by the "Adolf Messer Stiftung”, Bad Soden, Germany.

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  • Cited by (0)

    1

    Department of Urology and Pediatric Urology, University Medical Center Mainz, Mainz, Germany.

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