Inhibition of Colorectal Cancer Cell Survival by Paclitaxel Combined with Olaparib

Colorectal cancer is one of the main reasons for cancer-related mortality around the world and conventional chemotherapy approaches lead the clinical management of colorectal cancer treatment. Although many chemotherapeutics including paclitaxel have been effectively used to treat colorectal cancer patients, some tumour cells acquire drug resistance by increasing the regulation of various cellular mechanisms, primarily becoming addictive to some DNA repair activities. Preclinical evaluation of small-molecule inhibitors targeting various DNA damage response components is currently an active area of cancer research. This study aimed to assess the cytotoxic effect of paclitaxel combined with small-molecule PARP inhibitor olaparib in HCT116 colorectal cancer cells. We conducted clonogenic assays, showing that the paclitaxel and olaparib combination significantly inhibits the survival of colorectal cancer cells compared to paclitaxel or olaparib alone. By performing two distinct immunological methodology, Western blotting and immunofluorescence, we further demonstrated that olaparib intensifies the DNA damage accumulation induced by paclitaxel treatment. In conclusion, our preclinical study with HCT116 colorectal cancer cells suggests that olaparib may potentially serve as a combination partner to paclitaxel for effective colorectal cancer treatment. olaparibin


Introduction
Colorectal cancer is the third common type of malignancy worldwide, leading one of the major causes of cancer-related mortality [1]. Surgery is offered for colorectal cancer patients as the most effective treatment option and chemotherapeutical agents are mainly used to eliminate the remaining cancerous cells, repressing tumour growth and reducing recurrence of the disease [2]. Although widely-used anti-cancer agents have improved the prognosis of colorectal cancer in the clinic, their lack of selectivity and acquired resistance remain as one of the major obstacles of the treatment management [3]. Therefore, it is necessary to offer novel treatment strategies that eradicate cancer cells by targeting different cellular mechanisms at the same time.
Paclitaxel has been used as an anti-cancer agent in the treatment of various malignancies.
It binds to tubulin and stabilizes polymerized microtubules, arresting cell cycle at the G2/M checkpoint and consequently leading apoptotic cell death [4][5][6]. The single-agent treatment of high-dose paclitaxel is limited due to difficult-to-tolerate side effects such as peripheral neuropathy and hypersensitivity [7] and acquired paclitaxel resistance through numerous mechanisms including microtubule mutations prevents the long-term clinical success [8].
Considering these drawbacks of high-dose paclitaxel therapy, recent studies have revealed that low-dose paclitaxel application may promise better treatment approach for some malignancies owing to more tolerable toxicity and considerable reduction in drug resistance [9][10][11][12][13]. The cytotoxic effect of paclitaxel treatment was also evaluated in colorectal cancer cells [14,15]. Li et al. reported that the low-dose paclitaxel treatment inhibits cell cycle at G0/G1 phase in HCT116 and LOVO cells by modulating the expression of Myc and phosphorylated-c-Myc [15].
Theoretically, low-dose paclitaxel treatment is utilized to suppress the invasive progress and metastatic activities of cancer instead of eliminating neoplastic cells through apoptotic cell death as in the conventional approach. However, contrary research argues that long-term low-dose paclitaxel single-agent therapy may provoke adverse effects such as promoting metastasis [16]. Therefore, researchers have been studying to evaluate the potential of paclitaxel at a low toxic dose combined with small-molecule inhibitors in different experimental settings [17]. Taken together, a combination of paclitaxel and novel small-molecule inhibitors may potentially offer more effective and tolerable anti-cancer responses for the treatment of colorectal cancer patients.
DNA is subjected continual insults from endogenous and exogenous sources and key proteins involved in DNA damage response (DDR) pathway are required to monitor the integrity of genetic material and to activate cell cycle checkpoints once the damage induced, including poly ADP-ribose (PAR) polymerases (PARP) [18,19]. The PARP enzymes are classified under the group of ADP-ribosyltransferases (ARTs) and contribute to a variety of essential cellular processes including chromatin modification, cell cycle checkpoint regulation and the repair of DNA single-strand breaks (SSBs) [20,21]. The enzymatic activity of PARPs can be silenced by PARP inhibitors, some of those have been subjected to intense preclinical and clinical investigations for the treatment of certain cancer patients with BRCA1/2 mutations either alone or in combination with chemotherapy drugs [22]. PARP inhibitors olaparib, rucaparib and niraparib have been granted the USFDA approval for the treatment of patients with germline BRCA-mutant ovarian cancer [23]. Olaparib has recently been approved for germline BRCA-mutant metastatic breast [24] and pancreatic cancer treatments [25]. The effective use of PARP inhibitors in the management of colorectal cancer treatment has recently gained attention. Arena et al. demonstrated that PARP inhibitors have a potential to limit colorectal tumour growth and disease progression [26]. It has been also revealed that colorectal cancer cells become vulnerable to irinotecan therapy upon treatment of PARP inhibitors rucaparib [27] or niraparib [28]. Here, we hypothesized that the combination treatment of microtubule-targeting agent paclitaxel and PARP inhibitor olaparib would synergize the cytotoxic activities of these molecules, potentially offering more solid management of colorectal cancer treatment. We found that olaparib may intensify paclitaxel cytotoxicity through increasing the overall DNA damage burden in HCT116 colorectal cancer cells.

Cell culture conditions
HCT116 colorectal cancer cell line was kindly provided by Dr Alexander Hergovich (UCL, London, UK) and maintained in high glucose DMEM supplemented with 10% fetal bovine serum with 1% antibiotic (pen-strep). Cells were grown in humidity-saturated cell culture incubators at 37°C with 5% CO2. Stock solutions of olaparib, rucaparib and paclitaxel were prepared in DMSO and stored at -80°C. Drug/inhibitor treatments were performed as indicated in the corresponding figure legends.

Clonogenic survival assays
Clonogenic survival assays were performed as described in [29]. Briefly, 1500 of exponential phase cells were seeded in 6-cm plates and allowed to adhere for 24 hours, before being treated with olaparib or rucaparib with/out paclitaxel for 3 days. Cell medium was refreshed every 4 days until each colony has more than 50 cells (8-10 days). Colonies were first fixed with methanol/acidic acid (3:1) solution, then stained with 0.5% crystal violet dissolved in methanol for 15 minutes. After letting the plates air-dry overnight at room temperature, the number of colonies was counted per plate. The plating efficiencies of the corresponding controls were used to calculate the surviving fraction.

Western blotting
Protein lysates were resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk prepared in TBS-T (50 mM Tris, 150 mM NaCl, 0.5% Tween-20, pH 7.5) and then incubated with the corresponding antibody overnight. The protein-antibody complex was then probed by secondary antibodies conjugated with horseradish peroxidase (HRP) and finally subjected to ECL (Amersham) substrates for chemiluminescent detection. Densitometry analysis of Western blots was conducted using the NIH ImageJ.

Immunofluorescence microscopy
Immunofluorescence experiments were conducted as defined in [30]. Briefly, cells cultured on glass coverslips were fixed in 3%-paraformaldehyde (PFA) and 2%-sucrose solution for 20 minutes at room temperature, permeabilized for 2 minutes with 0.5% (vol/vol) Triton X-100 in PBS, washed in PBS, and then incubated with anti-gH2AX primary antibody (1:100) overnight at 4°C. Following the washing step, the coverslips were incubated with blocking buffer (10% goat serum) before being incubated with anti-rabbit Texas Red secondary antibody (1:100) and DAPI (1 μg/mL) for 2 hours in the dark at room temperature, washed with PBS, and mounted using Vectashield mounting medium (Vector Lab) in a microscope slide. Finally, the corners of the cover slides were sealed with nail polish and images were obtained with an Apotome fluorescence microscope (Zeiss) and Photoshop CS5 (Adobe Systems Inc.).

Statistical analysis
GraphPad Prism software was used to acquire graphics and statistical analyses. Data are displayed as mean ± SEM. The significance of differences between the means was one-tailed unpaired Student's t-test for colony survival assays and Mann-Whitney test for immunofluorescence analyses. Differences were considered statistically significant when pvalues were below 0.05 (*), 0.01 (**), 0.001 (***) or 0.0001 (****) for all experiments.

Paclitaxel and Olaparib Combination Inhibits HCT116 Cancer Cell Survival
As a widely used anti-neoplastic agent in the clinic [5], paclitaxel treatment causes tubulin polymerization, dysfunctional microtubule formation and cell cycle arrest, ultimately inducing cell death [4,6,31]. Here, the cytotoxic effect of paclitaxel alone or in combination with smallmolecule PARP inhibitors olaparib and rucaparib (Fig. 1A) were examined by in vitro clonogenic analysis. We employed HCT116 colorectal cancer cells characterized by microsatellite instability (MSI) [32], displaying inefficient mismatch repair and chemoresistant profile [33]. First, the cytotoxic capacity of paclitaxel single-agent treatment was evaluated in HCT116 cells. After  Next, we evaluated the combined effect of paclitaxel (1 nM) and olaparib treatment on the clonogenic capacity of HCT116 cells. Cells were treated with olaparib (0.5 and 1 µM) alone or in combination with paclitaxel (1 nM) for 3 days, then further incubated with the medium without any drug. Interestingly, the paclitaxel and olaparib combination treatment had significantly repressed the survival of HCT116 colorectal cancer cells compared to the relevant single-agent treatments ( Fig. 2A/B). This synergistic cytotoxicity was specific to HCT116 colorectal cancer cells since the combination treatment did not affect the clonogenic survival of U2OS osteosarcoma cells that were subjected to the same experimental procedures (Fig. 2C). To consolidate our findings, we further analyzed the cytotoxic activity of paclitaxel combined with rucaparib, another USFDA approved small-molecule PARP inhibitor, in HCT116 cancer cells. Cells were treated with rucaparib (0.1 and 0.5 µM) alone or in combination with paclitaxel (1 nM) for 3 days as detailed above. In line with previous results, we noticed a significant reduction in colony survival of HCT116 cells upon paclitaxel and rucaparib combination regimen compared to the relevant single-agent treatments (Fig. 3A/B). In summary, our clonogenic survival experiments revealed that HCT116 colorectal cancer cells become vulnerable to PARP inhibitor treatment when paclitaxel is included as a combination partner.

Olaparib treatment augments the Paclitaxel-induced DNA damage accumulation in HCT116 cells
After deciphering that HCT116 colorectal cancer cells have significantly impaired clonogenic capacity upon the paclitaxel and olaparib combination treatment, we further investigated the level of DNA damage induction in HCT116 cancer cell treated with paclitaxel and olaparib combined or alone. As a reliable DNA damage indicator [36], the phosphorylation status of H2AX (gH2AX-Ser139) was assessed by conducting Western blotting. HCT116 cells were treated with olaparib (1 µM) or paclitaxel (1 nM) as single agents or in combination for 24 hours. Following treatment, cells were incubated with the drug-free medium for additional 4 hours for the recovery before being subjected to Western blotting analysis (Fig. 4A). As presented in   representative images of immunodetection of gH2AX foci (red) in HCT116 cells exposed to beforementioned treatments for 24 hours. DNA is stained blue

Discussion
Cytotoxic chemotherapy is widely used for the management of many cancer treatments; however, de novo or acquired drug resistance, as well as side effects, limit the clinical success of anti-neoplastic agents. The main mechanism of action of the most chemotherapeutics is to bombard the genome with various types of DNA lesions up to a certain level that a cell decides to death [37]. This intensified DNA damaging pressure increases the genomic instability of tumour cells, provoking them to alter the regulation of various cellular pathways to survive including DNA damage response (DDR) and repair mechanisms. Therefore, the combination of conventional drugs with smart small-molecule inhibitors targeting DDR components would potentially offer better treatment responses. Unlike directly DNA damaging cytotoxic drugs, paclitaxel interferes with the disassembly of microtubules by promoting tubulin polymerization.
The formation of stable and dysfunctional microtubules causes the inhibition of cell cycle regulation and ultimately induces cell death [4][5][6]31]. In this study, the combination of low-dose paclitaxel with olaparib or rucaparib was evaluated for treating colorectal cancers. The combination treatment of HCT116 cells with paclitaxel and olaparib revealed a much stronger inhibitory effect on clonogenic formation compared with paclitaxel or olaparib alone. Rucaparib treatment with paclitaxel provided similar results in the clonogenic evaluation of HCT116 cells.
It has been shown that 1 nM paclitaxel treatment arrests the cell cycle at the G1 phase in HCT116 cells [12]. Considering PARP enzymes essential for the induction of G1 arrest [38], one can argue that PARP inhibition may potentially prevent the G1 arrest and therefore allow paclitaxel-treated cancer cells with dysfunctional microtubules towards the S phase, consequently causing more catastrophic DNA damage induction. Indeed, we revealed that HCT116 colorectal cancer cells acquired more DNA damage upon the combined paclitaxel and olaparib treatment compared to single-agent treatments. HCT116 colorectal cancer cells are deficient in mismatch repair and therefore characterized by microsatellite instability (MSI) [32]. Williams et al. demonstrated that the MSI status of colorectal cancer cells is dispensable for the response to PARP inhibitor niraparib, alone or in combination with DNA damaging agent irinotecan [28]. Therefore, further research is warranted to fully understand whether the genomic and proteomic profiles of HCT116 cells [32] are significant in this enhanced therapeutic response to paclitaxel and olaparib combined treatment. In addition, since paclitaxel can also cause DNA single-strand breaks (SSBs) [39][40][41], deficient SSB repair due to PARP inhibition may induce the accumulation of paclitaxel-induced SSBs which are eventually converted to disastrous DNA double-strand breaks (DSBs) during DNA replication [42]. Inefficient homologous recombination DSB repair mechanism owing to a possible BRCA2 mutation in HCT116 cells [32] may also explain why the paclitaxel-olaparib treatment induces more DNA damage and less clonogenic survival of HCT116 cells. Detailed mechanisms remain to be elucidated.

Conclusion
The efficacy of paclitaxel and olaparib, both alone and in combination, was assessed in HCT116 colorectal cancer cells. Our results suggest that the combination of paclitaxel and olaparib may need to be further studied to enhance the therapeutic responses of colorectal cancer patients.