Pirfenidone Sensitizes NCI-H460 Non-Small Cell Lung Cancer Cells to Paclitaxel and to a Combination of Paclitaxel with Carboplatin

Pirfenidone, an antifibrotic drug, has antitumor potential against different types of cancers. Our work explored whether pirfenidone sensitizes non-small cell lung cancer (NSCLC) cell lines to chemotherapeutic treatments. The cytotoxic effect of paclitaxel in combination with pirfenidone against three NSCLC cell lines (A549, NCI-H322 and NCI-H460) was evaluated using the sulforhodamine B assay. The effects of this combination on cell viability (trypan blue exclusion assay), proliferation (BrdU incorporation assay), cell cycle (flow cytometry following PI staining) and cell death (Annexin V-FITC detection assay and Western blot) were analyzed on the most sensitive cell line (NCI-H460). The cytotoxic effect of this drug combination was also evaluated against two non-tumorigenic cell lines (MCF-10A and MCF-12A). Finally, the ability of pirfenidone to sensitize NCI-H460 cells to a combination of paclitaxel plus carboplatin was assessed. The results demonstrated that pirfenidone sensitized NCI-H460 cells to paclitaxel treatment, reducing cell growth, viability and proliferation, inducing alterations in the cell cycle profile and causing an increase in the % of cell death. Remarkably, this combination did not increase cytotoxicity in non-tumorigenic cells. Importantly, pirfenidone also sensitized NCI-H460 cells to paclitaxel plus carboplatin. This work highlights the possibility of repurposing pirfenidone in combination with chemotherapy for the treatment of NSCLC.


Introduction
Despite recent breakthroughs in lung cancer diagnosis and management, this disease remains the second most commonly diagnosed cancer and the leading cause of cancerassociated death worldwide [1]. Lung cancer is a heterogenous group of diseases that can be classified into two major histologic types: small cell lung cancer (SCLC) and nonsmall cell lung cancer (NSCLC). Particularly, NSCLC accounts for roughly 85% of the cases and, depending on the tissue of origin, is categorized into three main histologic subtypes: adenocarcinoma (40%); squamous cell carcinoma (25-30%); and large cell carcinoma (5-10%) [2]. sensitizing effect of pirfenidone to other chemotherapeutic drugs currently being used in the clinic for NSCLC treatment has not been yet explored.
Our work intended to understand whether pirfenidone could sensitize NSCLC cells to treatment with paclitaxel, which is a chemotherapeutic drug currently used in the treatment of NSCLC patients. For that, the cell growth inhibitory effect of the combined treatment of paclitaxel with pirfenidone was evaluated in three human NSCLC cell linesthe adenocarcinoma A549 and NCI-H322 cell lines, and the large cell carcinoma NCI-H460 cell line. In addition, the effects of this combined drug treatment on cell viability, cell proliferation, cell cycle profile, cell death and on the expression levels of apoptotic-related proteins were further analyzed for the most sensitive cell line (NCI-H460). Importantly, the cytotoxic effect of this combined drug treatment on two non-tumorigenic cell lines, MCF-10A and MCF-12A, was also determined. Finally, the sensitizing effect of pirfenidone to the drug combination consisting of paclitaxel plus carboplatin, which is currently used in clinical practice, was also assessed in NCI-H460 cells.

The Combined Treatment of Paclitaxel with Pirfenidone Reduces the Growth of Three Human NSCLC Cell Lines
To investigate the tumor cell growth inhibitory activity of the combined treatment of paclitaxel with pirfenidone, the cytotoxic effect of each individual drug-paclitaxel or pirfenidone-was first evaluated, using the sulforhodamine B (SRB) assay. For that, three human NSCLC cell lines, A549, NCI-H322 and NCI-H460, were treated with five serial dilutions of each drug individually for 48 h, and the GI 50 concentrations (that cause 50% of cell growth inhibition) were assessed by an interpolation on the acquired dose-response curves (Supplementary Figure S1). The results, presented in Table 1, demonstrated that, as expected, all three NSCLC cell lines presented higher sensitivity to paclitaxel treatment than to pirfenidone. Indeed, as predicted, the GI 50 concentrations obtained for pirfenidone in the NSCLC cell lines were significantly higher than those obtained for the chemotherapeutic drug paclitaxel, as pirfenidone is not a drug commercially approved for cancer treatment. Table 1. GI 50 concentrations (µM) of paclitaxel and pirfenidone in three non-small cell lung cancer (NSCLC) cell lines.
Furthermore, the effect of the combined treatment of paclitaxel with pirfenidone on the % of cell growth on the three human NSCLC cell lines under study was evaluated using the SRB assay. The NCI-H460 cell line was treated for 48 h with drug combinations consisting of: (1) pirfenidone at 2 mM with increasing concentrations of paclitaxel (Figure 1(A1)); or (2) paclitaxel at 5.7 nM with increasing concentrations of pirfenidone (Figure 1(A2)). Results revealed that the combined treatment of paclitaxel 5.7 nM with pirfenidone 2.0 mM statistically significantly reduced the % of NCI-H460 cell growth, when compared with treatment with each drug individually. These results indicate that pirfenidone sensitizes NCI-H460 cells to paclitaxel treatment.
Next, these results were validated in two other NSCLC cell lines. The A549 cells were treated for 48 h with the drug combination consisting of paclitaxel 2.7 nM and pirfenidone 1.5 mM. The results presented in Figure 1B demonstrate that, even though the combined treatment presented a clear cytotoxic effect against this cancer cell line, this effect was not statistically significant when compared to treatment with each drug individually. The same outcome was observed when NCI-H322 cells were treated for 48 h with paclitaxel 5.7 nM The effect of the vehicle at the highest concentration tested was also analyzed. Results are presented as % of cell growth and are the mean ± SEM of at least three independent experiments. * or # p < 0.05, ** or ## p < 0.01 and ### p < 0.001.
Next, these results were validated in two other NSCLC cell lines. The A549 cells were treated for 48 h with the drug combination consisting of paclitaxel 2.7 nM and pirfenidone 1.5 mM. The results presented in Figure 1B demonstrate that, even though the combined treatment presented a clear cytotoxic effect against this cancer cell line, this effect was not statistically significant when compared to treatment with each drug individually. The same outcome was observed when NCI-H322 cells were treated for 48 h with paclitaxel 5.7 nM and pirfenidone 2 mM (concentrations that achieved the best results in the NCI-H460 cells). Therefore, we decided to proceed our studies in the NCI-H460 cells.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Reduces NCI-H460 Cell Viability and Proliferation
Considering the previous results, we then evaluated the effect of the combined treatment of paclitaxel with pirfenidone on the viability and proliferation of NCI-H460 cells, using the trypan blue exclusion assay and the bromodeoxyuridine (BrdU) incorporation assay, respectively. For that, NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel with 2.0 mM pirfenidone, as well as with each drug individually. The vehicle at the highest concentration tested was used as a negative control. Doxorubicin (50 nM) was used as a positive control. Figure 2 demonstrates that, as expected, doxorubicin statistically significantly reduced NCI-H460 cell viability. The NCI-H460 cells treated with either paclitaxel or pirfenidone statistically significantly reduced the % of viable cell number. Importantly, the combined treatment of paclitaxel with pirfenidone statistically significantly reduced NCI-H460 cell viability more efficiently than the treatment with each drug individually. The effect of the vehicle at the highest concentration tested was also analyzed. Results are presented as % of cell growth and are the mean ± SEM of at least three independent experiments. * or # p < 0.05, ** or ## p < 0.01 and ### p < 0.001.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Reduces NCI-H460 Cell Viability and Proliferation
Considering the previous results, we then evaluated the effect of the combined treatment of paclitaxel with pirfenidone on the viability and proliferation of NCI-H460 cells, using the trypan blue exclusion assay and the bromodeoxyuridine (BrdU) incorporation assay, respectively. For that, NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel with 2.0 mM pirfenidone, as well as with each drug individually. The vehicle at the highest concentration tested was used as a negative control. Doxorubicin (50 nM) was used as a positive control. Figure 2 demonstrates that, as expected, doxorubicin statistically significantly reduced NCI-H460 cell viability. The NCI-H460 cells treated with either paclitaxel or pirfenidone statistically significantly reduced the % of viable cell number. Importantly, the combined treatment of paclitaxel with pirfenidone statistically significantly reduced NCI-H460 cell viability more efficiently than the treatment with each drug individually.

Figure 2.
Effect of the combined treatment of paclitaxel with pirfenidone on the relative viable cell number of NCI-H460 cells, assessed by the trypan blue exclusion assay. Cells were treated for 48 h with 5.7 nM paclitaxel and 2 mM pirfenidone, either alone or in combination. The effect of the vehicle at the highest concentration tested in the drug treatments was also analyzed. Doxorubicin (50 nM) was used as a positive control. Results are the mean ± SEM of at least three independent experiments. ** p < 0.01, ### or +++ p < 0.001 and ++++ p < 0.0001.
Moreover, results from the BrdU incorporation assay ( Figure 3) demonstrated that paclitaxel and pirfenidone alone caused a statistically significant reduction in the % of NCI-H460 proliferating cells. A further decrease in the % of proliferating cells was found following treatment with the combination of paclitaxel with pirfenidone, when compared with paclitaxel alone.  Moreover, results from the BrdU incorporation assay ( Figure 3) demonstrated that paclitaxel and pirfenidone alone caused a statistically significant reduction in the % of NCI-H460 proliferating cells. A further decrease in the % of proliferating cells was found following treatment with the combination of paclitaxel with pirfenidone, when compared with paclitaxel alone.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Induces Major Alterations on the Cell Cycle Profile of NCI-H460 Cells
Taking into consideration the effect of the combined treatment of paclitaxel with pirfenidone on NCI-H460 cell growth, viability and proliferation, we then explored whether these effects could be related to alterations in the cell cycle profile, using flow cytometry following propidium iodide (PI) staining. The NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, with each drug alone, with the vehicle at the highest concentration tested (control) and with doxorubicin (50 nM, positive control). Results presented in Figure 4 revealed that, as described in the literature [37,38], doxorubicin caused major alterations in the cell cycle profile. Our results demonstrated that paclitaxel alone caused a statistically significant decrease in the % of cells in the G0/G1 phases of the cell cycle, accompanied by an increase in the sub-G1 cell population. Contrarily, a statistically significant increase in the % of cells in G0/G1, as well as a significant reduction in the % of cells in the S phase of the cell cycle, were found when NCI-H460 cells were treated with 2.0 mM pirfenidone. Regarding the combined treatment of paclitaxel with pirfenidone, our results showed a statistically significant reduction in the % of cells in the G2/M phases of the cell cycle, along with a prominent increase in the % of cells in the sub-G1 phase (suggestive of apoptosis), when compared with the effect of each drug alone. Thus, these results suggest that the combined treatment of paclitaxel with pirfenidone interferes with the cell cycle profile of NCI-H460 cells.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Induces Major Alterations on the Cell Cycle Profile of NCI-H460 Cells
Taking into consideration the effect of the combined treatment of paclitaxel with pirfenidone on NCI-H460 cell growth, viability and proliferation, we then explored whether these effects could be related to alterations in the cell cycle profile, using flow cytometry following propidium iodide (PI) staining. The NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, with each drug alone, with the vehicle at the highest concentration tested (control) and with doxorubicin (50 nM, positive control). Results presented in Figure 4 revealed that, as described in the literature [37,38], doxorubicin caused major alterations in the cell cycle profile. Our results demonstrated that paclitaxel alone caused a statistically significant decrease in the % of cells in the G0/G1 phases of the cell cycle, accompanied by an increase in the sub-G1 cell population. Contrarily, a statistically significant increase in the % of cells in G0/G1, as well as a significant reduction in the % of cells in the S phase of the cell cycle, were found when NCI-H460 cells were treated with 2.0 mM pirfenidone. Regarding the combined treatment of paclitaxel with pirfenidone, our results showed a statistically significant reduction in the % of cells in the G2/M phases of the cell cycle, along with a prominent increase in the % of cells in the sub-G1 phase (suggestive of apoptosis), when compared with the effect of each drug alone. Thus, these results suggest that the combined treatment of paclitaxel with pirfenidone interferes with the cell cycle profile of NCI-H460 cells.  was used as positive control. The effect of the vehicle at the highest concentration tested in the drug treatments was also analyzed. Results are the mean ± SEM of at least 3 independent experiments. *, # or + p < 0.05, ## or ++ p < 0.01 and +++ p < 0.001.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Increases NCI-H460 Cell Death
Since our findings pointed to the possibility that the combination of paclitaxel with pirfenidone induced NCI-H460 cell death (due to an increase in the sub-G1 phase of the cell cycle), we then evaluated the effect of this combined treatment on the levels of cell death, using Annexin V-FITC/PI labelling followed by flow cytometry analysis. For that, the NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, with each drug individually, with the vehicle at the highest concentration tested (control) and with 50 nM doxorubicin (positive control). The results ( Figure 5) demonstrated that both paclitaxel and pirfenidone alone increased NCI-H460 cell death. The combined treatment of 5.7 nM paclitaxel with 2.0 mM pirfenidone was more effective in increasing the % of NCI-H460 cell death, when compared to each individual treatment. These results confirm that the combination of paclitaxel with pirfenidone efficiently induces NCI-H460 cell death.

The Combined Treatment of Paclitaxel with Pirfenidone Efficiently Increases NCI-H460 Cell Death
Since our findings pointed to the possibility that the combination of paclitaxel with pirfenidone induced NCI-H460 cell death (due to an increase in the sub-G1 phase of the cell cycle), we then evaluated the effect of this combined treatment on the levels of cell death, using Annexin V-FITC/PI labelling followed by flow cytometry analysis. For that, the NCI-H460 cells were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, with each drug individually, with the vehicle at the highest concentration tested (control) and with 50 nM doxorubicin (positive control). The results ( Figure 5) demonstrated that both paclitaxel and pirfenidone alone increased NCI-H460 cell death. The combined treatment of 5.7 nM paclitaxel with 2.0 mM pirfenidone was more effective in increasing the % of NCI-H460 cell death, when compared to each individual treatment. These results confirm that the combination of paclitaxel with pirfenidone efficiently induces NCI-H460 cell death.

The Combined Treatment of Paclitaxel with Pirfenidone Causes Alterations in the Expression Levels of Apoptotic-Related Proteins
In order to further confirm the effect of the drug combination on NCI-H460 cell death, we then evaluated the effect of paclitaxel with pirfenidone, either alone or in combination, in the expression levels of some apoptotic-related proteins, such as PARP and caspase-3, by Western blot analysis. For that, NCI-H460 cells were treated for 48 h with the combined treatment (5.7 nM paclitaxel and 2.0 mM pirfenidone), with each drug alone and with the vehicle at the highest concentration tested in the drug treatments (control).
The results presented in Figure 6 show a prominent decrease in the expression levels of total poly (ADP-ribose) polymerase 1 (PARP-1), accompanied by an increase in the levels of cleaved PARP-1, when NCI-H460 cells were treated with the drug combination. Moreover, an increase in the expression levels of cleaved caspase-3 was observed following treatment with either paclitaxel alone or with the drug combination. These results corroborate our previous data, which demonstrated a significant increase in the % of NCI-H460 cell death after treatment with the combination of paclitaxel with pirfenidone.

The Combined Treatment of Paclitaxel with Pirfenidone Causes Alterations in the Expression Levels of Apoptotic-Related Proteins
In order to further confirm the effect of the drug combination on NCI-H460 cell death, we then evaluated the effect of paclitaxel with pirfenidone, either alone or in combination, in the expression levels of some apoptotic-related proteins, such as PARP and caspase-3, by Western blot analysis. For that, NCI-H460 cells were treated for 48 h with the combined treatment (5.7 nM paclitaxel and 2.0 mM pirfenidone), with each drug alone and with the vehicle at the highest concentration tested in the drug treatments (control).
The results presented in Figure 6 show a prominent decrease in the expression levels of total poly (ADP-ribose) polymerase 1 (PARP-1), accompanied by an increase in the levels of cleaved PARP-1, when NCI-H460 cells were treated with the drug combination. Moreover, an increase in the expression levels of cleaved caspase-3 was observed following treatment with either paclitaxel alone or with the drug combination. These results corroborate our previous data, which demonstrated a significant increase in the % of NCI-H460 cell death after treatment with the combination of paclitaxel with pirfenidone.

The Combined Treatment of Paclitaxel with Pirfenidone Does Not Cause More Cytotoxic Effect on Human Non-Tumorigenic Cells Than Paclitaxel Alone
Furthermore, we evaluated whether the combined treatment of paclitaxel with pirfenidone induced a cytotoxic effect against two human non-tumorigenic cell lines, MCF-10A and MCF-12A, using the SRB assay. For that, both cell lines were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, as well as with each drug alone. The effect of the vehicle at the highest concentration tested was also evaluated as a negative control. Our results (Figure 7) demonstrated that the drug combination consisting of paclitaxel with pirfenidone did not augment cytotoxicity to both non-tumorigenic cell lines, when compared to paclitaxel treatment alone.

Pirfenidone Sensitizes NCI-H460 Cells to the Combined Treatment of Paclitaxel Plus Carboplatin
Finally, we then analyzed the effect of pirfenidone in sensitizing NCI-H460 cells to the combined treatment of paclitaxel and carboplatin, by measuring the % of cell growth. For that, NCI-H460 cells were treated for 48 h with each drug individually-5.7 nM paclitaxel, 18.0 µM carboplatin and 2.0 mM pirfenidone, with the duplet (paclitaxel and carboplatin), with the triplet (paclitaxel and carboplatin plus pirfenidone) and with the

The Combined Treatment of Paclitaxel with Pirfenidone Does Not Cause More Cytotoxic Effect on Human Non-Tumorigenic Cells than Paclitaxel Alone
Furthermore, we evaluated whether the combined treatment of paclitaxel with pirfenidone induced a cytotoxic effect against two human non-tumorigenic cell lines, MCF-10A and MCF-12A, using the SRB assay. For that, both cell lines were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, as well as with each drug alone. The effect of the vehicle at the highest concentration tested was also evaluated as a negative control. Our results (Figure 7) demonstrated that the drug combination consisting of paclitaxel with pirfenidone did not augment cytotoxicity to both non-tumorigenic cell lines, when compared to paclitaxel treatment alone.

The Combined Treatment of Paclitaxel with Pirfenidone Does Not Cause More Cytotoxic Effect on Human Non-Tumorigenic Cells Than Paclitaxel Alone
Furthermore, we evaluated whether the combined treatment of paclitaxel with pirfenidone induced a cytotoxic effect against two human non-tumorigenic cell lines, MCF-10A and MCF-12A, using the SRB assay. For that, both cell lines were treated for 48 h with the combined treatment consisting of 5.7 nM paclitaxel and 2.0 mM pirfenidone, as well as with each drug alone. The effect of the vehicle at the highest concentration tested was also evaluated as a negative control. Our results (Figure 7) demonstrated that the drug combination consisting of paclitaxel with pirfenidone did not augment cytotoxicity to both non-tumorigenic cell lines, when compared to paclitaxel treatment alone.

Pirfenidone Sensitizes NCI-H460 Cells to the Combined Treatment of Paclitaxel plus Carboplatin
Finally, we then analyzed the effect of pirfenidone in sensitizing NCI-H460 cells to the combined treatment of paclitaxel and carboplatin, by measuring the % of cell growth. For that, NCI-H460 cells were treated for 48 h with each drug individually-5.7 nM paclitaxel, 18.0 µM carboplatin and 2.0 mM pirfenidone, with the duplet (paclitaxel and carboplatin), with the triplet (paclitaxel and carboplatin plus pirfenidone) and with the vehicle (at the highest concentration tested in the drug treatments). The obtained results (Figure 8) demonstrated that the triplet drug combination (paclitaxel and carboplatin plus pirfenidone) statistically significantly reduced the % of NCI-H460 cell growth, when compared to treatment with the duplet (paclitaxel and carboplatin). Therefore, our data showed that pirfenidone not only sensitized NCI-H460 cells to paclitaxel treatment, but also to paclitaxel plus carboplatin treatment, both regimens currently used in the clinical practice for NSCLC treatment. vehicle (at the highest concentration tested in the drug treatments). The obtained results ( Figure 8) demonstrated that the triplet drug combination (paclitaxel and carboplatin plus pirfenidone) statistically significantly reduced the % of NCI-H460 cell growth, when compared to treatment with the duplet (paclitaxel and carboplatin). Therefore, our data showed that pirfenidone not only sensitized NCI-H460 cells to paclitaxel treatment, but also to paclitaxel plus carboplatin treatment, both regimens currently used in the clinical practice for NSCLC treatment. Additionally, we also assessed the ability of pirfenidone to sensitize cells from two NSCLC cell lines (NCI-H460 and A549) to other chemotherapeutic drugs, such as etoposide and gemcitabine, which are also applied in the clinical practice for the treatment of NSCLC (Supplementary Table S1). Our results demonstrated that pirfenidone in combination with etoposide (Supplementary Figure S2) or gemcitabine (Supplementary Figure  S3) did not bring advantage over these chemotherapeutic drugs used alone. Moreover, the effect of the combined treatment of pirfenidone with etoposide plus carboplatin (Supplementary Figure S4), which is a duplet commonly applied for the treatment of lung large cell carcinoma, was also evaluated. However, no statistically significant difference was found when comparing this triplet drug combination with the duplet drug combination consisting of etoposide plus carboplatin.

Discussion
Lung cancer is the world's deadliest malignancy, and NSCLC is the most frequently diagnosed histologic type [1,2]. Despite recent advances in lung cancer treatment, with the introduction of immune and targeted therapies, the use of these therapeutic modalities as single systemic agent regimens is not suitable for a large number of patients, and chemotherapy remains critical for clinical management [8,10]. Thus, the development of new and more effective therapeutic options such as drug combinations is strictly necessary. Over the past few years, drug repurposing has emerged in oncology as an appealing strategy to identify antitumor potential in drugs already approved or under investigation for the treatment of other diseases [39]. Pirfenidone, a drug approved for the treatment of Additionally, we also assessed the ability of pirfenidone to sensitize cells from two NSCLC cell lines (NCI-H460 and A549) to other chemotherapeutic drugs, such as etoposide and gemcitabine, which are also applied in the clinical practice for the treatment of NSCLC (Supplementary Table S1). Our results demonstrated that pirfenidone in combination with etoposide (Supplementary Figure S2) or gemcitabine (Supplementary Figure S3) did not bring advantage over these chemotherapeutic drugs used alone. Moreover, the effect of the combined treatment of pirfenidone with etoposide plus carboplatin (Supplementary Figure S4), which is a duplet commonly applied for the treatment of lung large cell carcinoma, was also evaluated. However, no statistically significant difference was found when comparing this triplet drug combination with the duplet drug combination consisting of etoposide plus carboplatin.

Discussion
Lung cancer is the world's deadliest malignancy, and NSCLC is the most frequently diagnosed histologic type [1,2]. Despite recent advances in lung cancer treatment, with the introduction of immune and targeted therapies, the use of these therapeutic modalities as single systemic agent regimens is not suitable for a large number of patients, and chemotherapy remains critical for clinical management [8,10]. Thus, the development of new and more effective therapeutic options such as drug combinations is strictly necessary. Over the past few years, drug repurposing has emerged in oncology as an appealing strategy to identify antitumor potential in drugs already approved or under investigation for the treatment of other diseases [39]. Pirfenidone, a drug approved for the treatment of idiopathic pulmonary fibrosis, has been demonstrated to have antitumor potential as well as to sensitize some tumor models to chemotherapy [22][23][24][25][26][27][28][29][30][31][32]34,35,40]. Importantly, a retrospective study demonstrated that patients with idiopathic pulmonary fibrosis treated with pirfenidone had lower incidences of lung cancer [41]. Therefore, due to the previously described antitumor effect of pirfenidone, our work aimed to evaluate the ability of pir-fenidone to sensitize NSCLC cell lines to paclitaxel, a chemotherapeutic drug currently used in clinical practice, with the ultimate aim of contributing to pre-clinical data for the possibility of repurposing pirfenidone in combination with paclitaxel-based regimens for the treatment of NSCLC.
Our data clearly revealed that the combined treatment of paclitaxel with pirfenidone reduced NCI-H460 cell growth and viability more effectively than treatment with each drug individually, suggesting that pirfenidone sensitizes NCI-H460 cells to paclitaxel treatment. Moreover, this combined drug treatment caused major alterations on the cell cycle profile by reducing the % of NCI-H460 cells in the G2/M phases and increasing the % of cells in the sub-G1 phase. These results suggest that this combined drug treatment caused a decrease in cell proliferation and an increase in cell death. Indeed, these results were further corroborated, since a reduction in cell proliferation, an increase in cell death by apoptosis, and a decrease in the levels of total PARP-1, accompanied by an increase in the levels of cleaved PARP-1 and caspase-3, were observed when NCI-H460 cells were treated with the combination consisting of pirfenidone with paclitaxel.
Our findings are consistent with those reported by Mediavilla-Varela M. et al. (2016), which described a significant reduction in proliferation, accompanied by an increase in cell death, when several NSCLC cell lines were treated with the combined treatment consisting of pirfenidone with cisplatin [35]. Therefore, our data together with the literature suggests that pirfenidone sensitizes NSCLC cell lines to platinum-based chemotherapeutic regimens.
Furthermore, our data corroborates the previously described antitumor potential of pirfenidone treatment alone. Indeed, our work demonstrated that pirfenidone reduces NCI-H460 cell growth, viability and proliferation, and induces a G0/G1 cell cycle arrest. Likewise, in 2019, Ishii K. et al. [23], Usugi E. et al. [24] and Marwitz S. et al. [30] demonstrated the same effects for pirfenidone treatment in prostate, pancreatic and NSCLC cancer models, respectively. In agreement with our results, these authors also found an increase in the % of cells in the G0/G1 phases of the cell cycle accompanied by a reduction in the % of cells in the S phase following pirfenidone treatment alone.
Importantly, we demonstrated for the first time that the combined treatment of paclitaxel with pirfenidone did not cause additional cytotoxicity against human non-tumorigenic cell lines, when compared with the chemotherapeutic drug used alone. In addition, our work demonstrated that pirfenidone sensitizes NCI-H460 cells to the combined treatment of paclitaxel with carboplatin, which is a chemotherapy duplet currently used in clinical practice for the treatment of NSCLC patients.
Taken together, our findings provide strong and innovative pre-clinical data to support the possibility of carrying out clinical studies to verify the effect of repurposing pirfenidone in combination with paclitaxel or with paclitaxel plus carboplatin, for the perioperative treatment of NSCLC. To our knowledge, this is the first study exploring the possibility of repurposing pirfenidone in combination with combinatorial platinum-based regimens in vitro. Nevertheless, further pre-clinical studies using complex biological models (e.g., xenografted mice models) must be carried out, to gather enough pre-clinical data to support a clinical trial.

Drug Treatments
To determine the GI 50 concentration of each drug, the NSCLC cell lines were treated for 48 h with: (a) five serial dilutions of the tested drug; (b) vehicle (at the higher concentration used); and (c) medium alone. Carboplatin was tested at concentrations ranging from 6.3 to 100.0 µM; etoposide was tested at concentrations ranging from 0.2 to 10.0 µM; gemcitabine was tested at concentrations ranging from 1.9 × 10 −3 to 0.1 µM; paclitaxel was tested at concentrations ranging from 4.0 × 10 −4 to 7.0×10 −2 µM; and pirfenidone was tested at concentrations ranging from 313.0 to 5.0 × 10 3 µM.
To evaluate the cytotoxic effect of the combined drug treatments, the NSCLC cell line NCI-H460 was treated for 48 h with two distinct experimental designs: (a) combined treatment of five serial dilutions of etoposide, gemcitabine or paclitaxel with a selected concentration of pirfenidone; or (b) combined treatment of five serial dilutions of pirfenidone with a selected concentration of etoposide, gemcitabine or paclitaxel. The A549 cell line was treated with drug combinations consisting of etoposide, gemcitabine or paclitaxel and a selected concentration of pirfenidone. The concentrations of paclitaxel and pirfenidone that produced the best results for the NCI-H460 cell line were tested in the NCI-H322 cell line. The cytotoxic effect of the combined treatment of 5.7 nM paclitaxel with 2.0 mM pirfenidone against human non-tumorigenic cell lines, MCF-10A and MCF-12A, was evaluated. In all experimental designs, medium alone (blank) and vehicle of the drugs at the highest concentration tested were used as negative controls.
For the following assays, cells were treated for 48 h with: (a) the concentration of each drug alone; (b) the combined drug treatment; (c) vehicle (at the higher concentration tested in drug treatments); and (d) medium alone (blank).

Cell Viability-Trypan Blue Exclusion Assay
Cell number and viability were evaluated using the trypan blue exclusion assay. After drug treatments, cell suspensions were mixed with 0.2% (v/v) trypan blue dye (Merck Life Science, Darmstadt, Germany; T8154), which distinguishes viable (bright) from non-viable cells (blue ones), at a ratio of 1:1, and the cell number was counted using a hemocytometer (Neubauer Chamber).

Cell Growth Inhibition-Sulforhodamine B (SRB) Assay
To determine the concentration of each drug that caused 50% of cell growth inhibition (GI 50 ) and to assess the cytotoxic effect of the combined drug treatments, the sulforhodamine B (SRB) assay was performed, according to the described protocol [43]. For that, NSCLC cells were plated in 96-well plates at a previously determined optimal cell concentration (5 × 10 4 cells/mL) and incubated for 24 h. Then, cells were exposed to the desired drug treatments for 48 h. All studies were conducted in two distinct plates: one to be analyzed at the time of drug treatment (T0) and another to be analyzed 48 h later (T48).
Following a 48-h incubation, cells were fixed with 10% (w/v) ice-cold trichloroacetic acid (TCA; Merck Life Science, Darmstadt, Germany; T0699) for at least 1 h at 4 • C. After washing cells with distilled water and allowing them to air-dry at room temperature, cells were stained with 0.4% (w/v) SRB (Merck Life Science, Darmstadt, Germany; S9012) in 1% (v/v) acetic acid (Merck Life Science, Darmstadt, Germany; T0699) for 30 min, washed with 1% (v/v) acetic acid to remove the unbound dye and allowed to air-dry at room temperature. The bound SRB was then solubilized with 10 mM Tris base solution in water (Merck Life Science, Darmstadt, Germany; T6066) and absorbance was measured at 510 nm in a multi plate reader (Synergy TM Mx, Biotek Instruments Inc., Winooski, VT, USA), recurring to the Gen5 TM software.