Multimodality care of locally advanced rectal cancer includes chemotherapy, radiation, and surgery to achieve better local control and offer patients the best chance for long-term survival [1, 2]. Radiotherapy is most frequently administered preoperatively in the form of chemoradiation (CRT), which decreases local recurrence [3, 4]. However, response among patients varies greatly, with some tumors achieving a pathologic complete response (pCR, i.e., no viable tumor cells are present in the surgical specimen), while others demonstrate minimal treatment effect. Our group and others have shown that enhanced response to neoadjuvant therapy is associated with improved oncological outcomes [5,6,7]. Thus, it is reasonable to search for new approaches or adjuncts that can increase rectal cancer radiation sensitivity and improve outcomes.

The antineoplastic properties of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, commonly known as statins and prescribed for their serum cholesterol lowering effects, have been the focus of multiple studies over the past two decades [8, 9]. Population-based studies have indicated that patients on statin therapy may have a lower cancer risk in general [10], and also specifically colorectal cancer [11]. We previously observed and reported that patients with rectal cancer who were on statin therapy while receiving preoperative CRT had increased rates of pCR and improved overall response to therapy [12]. Similar findings have also been reported by other groups [13], underscoring the potential of statins as radiation-sensitizer agents in this setting.

While the results of the aforementioned retrospective studies are encouraging, prospective trials are necessary prior to the inclusion of statins in rectal cancer treatment protocols. In preparation for these trials, it is crucial to obtain preclinical data evaluating the effects of statins in combination with radiation on colorectal cancer (CRC) cells, and confirming the biological rationale behind this therapeutic regimen. The purpose of this study was to investigate the effects of simvastatin, as a representative member of the statin family of compounds, on colorectal cancer cell lines and explore its potential as a radiation-sensitizer in vitro.

Materials and methods

Culture of colorectal cancer cell lines

The commercially available colon adenocarcinoma cell lines SW480 and DLD1 and rectal adenocarcinoma cell lines HRT18 and SW837 were obtained from the American Type Culture Collection. Cell lines were maintained in DMEM medium supplemented with 10% fetal bovine serum and penicillin–streptomycin, with the exception of HRT18, which was maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin–streptomycin. Where indicated, cells were irradiated using a JL Shepherd Mark I 137Cs irradiator (San Fernando, CA).

Colony formation assay

Three thousand cells per well were plated in triplicate in a 6-well plate and treated with vehicle (DMSO) or simvastatin (1 or 2 µM), followed by irradiation (2 Gy) in the respective treatment arm 2 h later. Seven days later, upon visible colony formation, cell colonies were fixed, stained with a 0.1% crystal violet in 20% methanol solution, and counted.

Cell viability assay

Two hundred and fifty cells per well were plated in quadruplicate into 96-well plates containing the appropriate growth medium. The next day, vehicle (DMSO) or Simvastatin (1, 2, or 5 µM) was added to each well, followed by irradiation (2 Gy) in the respective treatment arm 2 h later. ATP levels were assessed via a CellTiter-Glo assay (Promega, Fitchburg, WI) on days 0, 3, 5, and 7 using a luminometer (Perkin-Elmer).

Immunoblotting assay

Cell protein extracts were obtained using lysis buffer, and protein concentration was determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein (50 mg) from each treatment condition were separated using SDS polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membranes were blocked in 5% milk solution for 1 h at room temperature. Rabbit anti-human antibodies were used to detect p44/42 MAPK (ERK1/2) (Cell Signaling, Danvers, MA, #9102, 1:500 dilution) and phospho-p44/42 MAPK (pERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA, #9101, 1:500 dilution). Mouse anti-human antibodies were used to detect cleaved caspase 3 (Cell Signaling, Danvers, MA, #9661, 1:500 dilution), and, as a loading control, b-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-47778, 1:2000 dilution). Horseradish peroxidase (HRP)-conjugated goat anti-mouse (for cleaved caspase 3 and b-actin; Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-2005, 1:2000 dilution) and goat anti-rabbit (for phospho-p44/42 MAPK and p44/42 MAPK, Santa Cruz Biotechnology Inc., Santa Cruz, CA, sc-2004, 1:2000 dilution) secondary antibodies were incubated for 1 h at room temperature and then the ECL system was used for protein detection (SuperSignal West Pico, ThermoFisher Scientific, Waltham, MA).

Statistical analyses

Statistical significance was calculated using a two-way Student’s t test on GraphPad Prism (GraphPad Software Inc., La Jolla, CA). Two-way p values less than 0.05 were considered statistically significant. Data are represented as the mean ± standard deviation.

Results

Simvastatin enhances the effects of radiation on CRC cells

Initially, we investigated the inhibitory effects of simvastatin, combined with radiation, on the growth of CRC cells. The combination of radiation and simvastatin was the most effective at inhibiting colony-forming ability of all four CRC lines, both compared to radiation alone (SW837, p < 0.05; SW480, p < 0.001; HRT18, p < 0.01; DLD1, p < 0.001), and compared to simvastatin alone (SW837, p < 0.05; SW480, p < 0.01; HRT18, p < 0.05; DLD1, p < 0.01) (Fig. 1A). These effects did not vary between the two more radiosensitive colon adenocarcinoma-derived lines (DLD1 and SW480) and the two relatively more radioresistant rectal adenocarcinoma-derived lines (SW837 and HRT18). In addition, the effects of simvastatin in this combination therapy were dose dependent, with increased concentrations resulting in more potentiated inhibitory effects.

Fig. 1
figure 1

Simvastatin enhances the effects of radiation on CRC cells. The inhibitory effects of simvastatin and radiation on 4 colorectal cancer cell lines were assessed by measuring the colony-forming capacity of these cells 7 days after treatment (A) and the cell viability (Cell Titer-Glo assay) 5 days after treatment (B) and as a time-course through 7 days after treatment (C). Cells were treated on day 0, with irradiation occurring 2 h after simvastatin was added to the media where indicated. p < 0.05, **p < 0.01, ***p < 0.001. Student’s t test was used to assess statistical significance

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The efficacy of the combination therapy on CRC cells was further confirmed with ATP-based viability studies. Cells treated with radiation and simvastatin had decreased viability 5 days after the treatment compared to radiation alone (SW837, p < 0.001; SW480, p < 0.001; HRT18, p < 0.05; DLD1, p < 0.001) or simvastatin alone (SW837, p < 0.01; SW480, p < 0.01; HRT18, p < 0.05; DLD1, p < 0.01), demonstrating again a dose-dependent effect of simvastatin (Fig. 1B). Results in 7-day experiments demonstrated a sustained effect, as there was no evidence of late recovery of cell viability. Again, combination therapy produced enhanced results compared to radiation alone (SW837, p < 0.001; HRT18, p < 0.01), or simvastatin alone (SW837, p < 0.001; HRT18, p < 0.01) (Fig. 1C).

The effects of simvastatin on the radiation sensitivity of CRC cells are dependent on GGPP depletion

The antineoplastic actions of statins are believed to be mediated through the depletion of isoprenoid molecules (cholesterol precursors downstream of HMG-CoA reductase) and the subsequent inhibition of isoprenylation of key cell cycle regulatory proteins such as RAS [14]. One of these molecules that have been directly linked with the effect of statins on neoplasia is geranyl-geranyl pyrophosphate (GGPP). To evaluate the dependency of the radiosensitizing effects of simvastatin on CRC cells on the depletion of GGPP, we performed the same viability experiments in the presence of exogenous GGPP in the culture media. As anticipated, the presence of exogenous GGPP largely negated the radiosensitizing effects of simvastatin (Fig. 2).

Fig. 2
figure 2

The effects of simvastatin on the radiation sensitivity of CRC cells are dependent on GGPP depletion. The ability of geranyl-geranyl pyrophosphate (GGPP) to rescue irradiated colorectal cancer cells from the effects of simvastatin was assessed by measuring the cell viability (Cell Titer-Glo assay) as a time-course through 7 days after treatment. Cells were treated on day 0, with irradiation occurring 2 h after simvastatin and GGPP were added to the media where indicated. p < 0.05, **p < 0.01, ***p < 0.001. Student’s t test was used to assess statistical significance

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Simvastatin inhibits ERK1/2 phosphorylation and increases apoptosis in irradiated CRC cells

To investigate the molecular effects of the combined treatment on CRC cells, we performed protein analyses of the treated cells. Cells from each condition were harvested 24 h after treatment—in irradiated cells, both colonic (SW480) and rectal (SW837), simvastatin treatment inhibited phosphorylation of ERK1/2, a downstream target of activated RAS, in a dose-dependent manner, while the total levels of ERK1/2 remained stable (Fig. 3). In addition, the combined treatment resulted in increased levels of cleaved caspase 3, indicating greater apoptotic activity in the cells treated with radiation and simvastatin together (Fig. 3).

Fig. 3
figure 3

Simvastatin inhibits ERK1/2 phosphorylation and increases apoptosis in irradiated CRC cells. The phosphorylation status of ERK1/2 and the presence of cleaved caspase 3 were evaluated in protein extracts from SW480 and SW837 cells 24 h after they treated with 1 or 5 μM of simvastatin as indicated, followed 2 Gy radiation. B-actin was used as internal control

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Discussion

The current study demonstrates that simvastatin inhibits the growth of colorectal cancer cells in vitro and increases their radiation sensitivity in a dose-dependent manner. The effects of simvastatin were present in both cell lines of colonic origin (SW480 and DLD1), which are relatively radiosensitive, and of rectal origin which are relatively radioresistant (HRT18 and SW837). In addition, simvastatin appeared to inhibit the viability and growth of irradiated CRC cells via the depletion of GGPP, a known cofactor of RAS, as the addition of exogenous GGPP to the cell media rescued the cells from these effects. Finally, as our focused pathway analysis demonstrated, simvastatin treatment was associated with decreased phosphorylation of ERK1/2 in these cells and increased apoptosis. Taken together with the known function of GGPP as a RAS cofactor, these data suggest a mechanistic role for the EGFR-RAS-ERK1/2 pathway underlying the statin effect on CRC radiosensitivity. These findings offer biological rationale of our clinical observation that simvastatin is associated with improved response to radiation in patients with locally advanced rectal cancer.

Our findings are in agreement with prior reports in the literature on the effects of statins on CRC cells in vitro and with our prior clinical observations. Statins have been shown to independently inhibit CRC cells, though most prior studies used doses higher than those of the present study [9, 15]. In our study, concentrations as low as 1 μM, which are comparable with the tissue concentrations achieved with oral statin therapy for hypercholesterolemia, successfully inhibited the growth of colorectal cancer cells and enhanced the effects of radiation. More importantly, statins have been shown to synergize with other clinically used cytotoxic or biologic therapies to improve response rates. The effects of the anti-VEGF agent bevacizumab [16], the anti-EGFR agent cetuximab [17, 18], and the chemotherapeutic 5-fluorouracil (5-FU) appear to be more potentiated in the presence of statins in vitro [8, 19]. The synergistic effect of statins and radiation is relatively less clear, but associations have been observed in other malignancies, such as breast cancer [20] and lung cancer [21]. Our results fit well within this background and support the notion that HMG-CoA metabolites are crucial in helping neoplastic cells overcome cytotoxic treatment. Furthermore, in accordance with the most widely supported antineoplastic mechanism of action for statins [22, 23], we found that the radiosensitizing effects of statins were dependent on the depletion of GGPP and, at least partially, involved the inhibition of ERK1/2 activation, downstream of RAS.

This study offers biological justification to pursue further research, both translational and clinical, on the role of statins as radiation sensitizing agents in rectal cancer. The enhanced efficacy of simvastatin combined with radiation on CRC cells in vitro validates the previously reported clinical observations and serves as basis for more detailed, mechanism-focused laboratory studies, as well as preclinical animal studies and clinical trials. Considering the oncologic impact of response to neoadjuvant (chemo-) radiation [7], as well as the potential for patients with complete response to forego surgery in non-operative active surveillance protocols (“watch & wait”), any adjunct therapy that enhances rectal cancer CRT response could significantly influence patient care. Statins are particularly attractive in this setting as they are relatively cheap, have a favorable safety profile, and have been used in clinical practice for decades.

It is important to interpret this study in the context of its limitations. The cell lines used here were immortalized, commercially available lines. Even though these have advantages such as reproducibility and comparability within the literature, they may not be as representative of in vivo tumor biology. In addition, while this study offers evidence that the inhibitory effects of simvastatin combined with radiation on CRC cells are greater than either treatment alone, the exact mechanism behind this phenomenon is not completely delineated. We hypothesized and confirmed a role for the depletion of GGPP and the decrease in ERK1/2 phosphorylation; however, it is likely that multiple pathways are implicated. Further mechanistic studies to delineate these pathways are beyond the scope of the present study and are the focus of future research. Finally, even though the in vitro data reported here are promising, preclinical animal models investigating the effects of statins and radiation on patient-derived rectal cancer xenografts would be the logical next step before proceeding to clinical trials. Therefore, this study is meant to serve as a proof-of-concept project for future research in this topic.

In conclusion, treatment with simvastatin hindered CRC cell viability and enhanced radiation sensitivity in vitro. These effects may be tied to the depletion of GGPP and the decreased phosphorylation of ERK1/2, suggesting a prominent role for the EGFR-RAS-ERK1/2 pathway through which statin enhances radiation sensitivity. Based on these findings, mechanistic and in vivo studies are in progress, with the ultimate goal of paving the way for a clinical trial.