A Histone Deacetylase Inhibitor Manifests Synergistic Interaction with Artesunate by Suppressing DNA Repair Activity

: Artesunate (ART), a plant based semi-synthetic antimalarial drug, is emerging as a new class of effective cancer chemotherapeutics. However, the dosage of ART required to have an anti-cancer effect on cancer cells is greater than that needed to exterminate malarial parasites. The goal of this study was to develop an effective combination therapy to reduce the dose-dependent side effects of ART both in vitro and in vivo. In our study, 4-phenylbutyrate (4-PB), a histone deacetylase inhibitor (HDAC), exhibited signiﬁcant synergistic induction of apoptosis in MCF-7 cells in combination with ART. The IC 50 of ART decreased signiﬁcantly from 55.56 ± 5.21 µ M to 24.71 ± 3.44 µ M in MCF-7 cells. ART treatment increased cellular oxidative stress, and the resulting generation of intracellular reactive oxygen species (ROS) caused extensive DNA damage in the cell. The extent of ROS production and cell cycle arrest were further enhanced by 4-PB treatment. In further investigation, we found that 4-PB attenuated mRNA expression of crucial DNA damage response (DDR) elements of the nonhomologous end-joining (NHEJ) pathway, consequently enhancing the DNA damaging effect of ART. Furthermore, the combination therapy resulted in improvement in the life expectancy of the treated mice and a prominent reduction in tumour volume without interfering with the normal biochemical, haematological and histological parameters of the mice. Overall, our study revealed a novel combination therapy in which 4-PB potentiated the cytotoxicity of ART synergistically and provided a promising combination drug for effective cancer therapy.


Introduction
In cellular oncogenesis, aberrant activity of histone deacetylases (HDACs) and histone acetyltransferases (HATs) has been observed in various instances. Much evidence suggests that the inactivation of HATs and abnormal HDACs expression results in the suppression of several antiproliferative genes in cancer cells, which further mediate tumour onset or progression [1]. Apart from regulating acetylation of histones, HDACs also participate in the modifications of various non-histone proteins, including repression of several transcription factors, chaperones, and signalling molecules, which may contribute to cancer cell survival [2].
HDACi modulates chromatin structure, resulting in the relaxation of chromatin and making it easier for transcription factors to load onto DNA [3]. This further regulates the expression of various tumour related genes involved in cell cycle control and apoptosis of cancer cells [4]. Histone deacetylase inhibitors (HDACi) have been known to inhibit cancer cell progression and cause programmed cancer cell death, both in vivo and in several pre-clinical models. Various HDACi have shown their potential as anti-cancer drugs, particularly in combination with other chemotherapeutic drugs in clinical studies [5].

Chemicals and Reagents
Chemicals, reagents, and kits used as a part of the accompanying investigations were obtained from Sigma-Aldrich, unless specified.

Cell Culture and Drug Treatment
MDA-MB-231 (human breast adenocarcinoma) and MCF-7 (human breast adenocarcinoma) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India. The cell line was routinely maintained in 10% fetal bovine serum (FBS) supplemented with Dulbecco's modified Eagle's medium (DMEM high glucose) containing 1% penicillin/streptomycin (100 U/mL; 0.1 mg/mL). The cells were grown in humidified air containing 5% CO 2 at 37 • C. 4-PB was freshly prepared as a 100 mM stock in PBS before each use. ART was resuspended in DMSO as a 100 mM stock solution.

Assessment of Cell Viability
The cell cytotoxicity activities of 4-PB and ART were assessed by the MTT assay (HiMedia, Mumbai, India) as previously described [14]. Briefly, 7 × 10 3 cells were seeded in 96-well plate in triplicates (n = 4). After 24 h of seeding, the cells were treated with different doses of drugs and combinations (keeping 5 mM of 4-PB constant) for 48 h. Fractional cell survival was assessed by taking absorbance at 570 nm (Infinite ® 200 PRO, Tecan, Switzerland) and normalising the background measurement at 650 nm. The viability of the untreated cells (or control group) was normalised at 100%.

HDAC Inhibition Assay
4-PB-induced HDAC inhibition was calculated using a fluorometric HDAC Activity Assay Kit. Briefly, cells were incubated with 5 mM of 4-PB and/or 50 µM ART for 4 h. The cells were then lysed in a solution containing protease inhibitor cocktail, 0.1% Triton X-100, 150 mM NaCl, and 50 mM HEPES. Following the manufacturer's instructions, the resulting cell lysates were processed using a sonicator. A plate reader (Infinite ® 200 PRO, Tecan, Switzerland) was used to measure the amount of fluorescence, with excitation at 350 nm and emission occurring at 440 nm.

Propidium Iodide (PI) Apoptosis Assay
The apoptotic population of the cells were analysed using the protocol described earlier [15]. MCF-7 cells were treated with 4-PB and/or ART for 48 h. The cells were collected, fixed in 70% ethanol, and processed in accordance with the aforementioned technique when the treatment period was over. After that, the cells were examined using a Beckman Coulter CytoFLEX flow cytometer with a 488 nm laser line as the excitation source. The PE-A channel was used to measure the red fluorescence of PI.

Fluoremetric Analyses of Apoptotic Cells Using PE Annexin V and 7-AAD
Following treatment with 5 mM 4-PB and/or 50 µM ART, the various apoptotic cell populations were counted using PE (phycoerythrin), Annexin V, and 7-AAD (7-aminoactinomycin D). Briefly, MCF-7 cells were treated with ART and/or 4-PB for 48 h. Following the manufacturer's instructions, the cells were harvested and stained with PE Annexin V and 7-AAD (BD Biosciences). The degree of apoptosis in the cells was examined using a flow cytometer (CytoFLEX, Beckman Coulter) as described earlier [16].

Combination Index (CI) Curve and Isobologram Analyses
The data generated in the cell viability experiment were analysed in COMPUSYN software to study the type of interaction between 4-PB and ART. The normalized isobologram generated by the software, based on Median-Effect Equation (Chou) and the Combination Index Theorem (Chou-Talalay), were then analysed. The CI was used to express antagonism (CI > 1), additivity (CI = 1) or synergism (CI < 1). The analyses were repeated up to four times. The average effect values (fa's) were used to plot Fa-Log (CI) curve.

Intracellular ROS Measurement
The levels of ROS generated after treatment with drugs were assessed using the dichlorodihydrofluorescein diacetate (DCF-DA) method [17]. Briefly, cells were treated with the 4-PB and/or ART for 6 h in the presence or absence of 500 µM of N-acetylcysteine (NAC) in the medium. Further, the cells were washed multiple times with PBS and incubated in serum-free medium containing 1 µM of DCFH-DA for 30 min at 37 • C. The data were then acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analysed using WinList 3D 9.0.1 software (Verity Software House, Topsham, ME, USA).

Cell Cycle Analysis
The cell cycle analysis was performed as described previously [18]. MCF-7 cells were synchronised by serum starvation for 24 h. Further, the cells were treated with 5 mM 4-PB and/or 50 µM ART for 36 h. After the completion of this treatment, the cells were fixed using 70 % ethanol and stained with 25 µM propidium iodide stain following RNAse (10 µg/mL) and 0.1% triton X-100 treatment. The data were acquired using a CytoFLEX flow cytometer (Beckman Coulter) and analysed using ModFit LT software (Verity Software House).

Relative mRNA Expression Analysis
Quantitative real-time PCR (qPCR) was performed to estimate the fold change in the expression of Ku70, Ku80, XRCC4, and DNA-PKcs using SYBR Green as a reporter dye (iTaq Universal SYBR Green Supermix, Bio-Rad, Hercules, CA, USA) in Rotor-Gene Q (Qiagen, Hilden, Germany). β-actin was taken as the endogenous control. The ∆∆Ct method was used to calculate the relative mRNA expression of Ku70, Ku80, XRCC4, and DNA-PKcs in LinRegPCR software.

In Vivo Study
For the in vivo study, male Swiss albino mice four months old weighing 22-25 g were purchased from Chakraborty enterprise (1443/PO/b/11/CPCSEA), Kolkata, India. All the animals were acclimatised to the laboratory environment at the Central Animal Facility, Institute of Advanced Study in Science and Technology (IASST), Guwahati, Assam, before conducting experiments. Animals were kept in polypropylene cages and maintained at 22 ± 2 • C and 60-70% relative humidity with a 12 h light-dark cycle. Throughout the experimental period, the animals were fed with a standard rodent pellet diet (Provimi Animal Nutrition India Pvt. Ltd., Bengaluru, KA, India). All the protocols were approved by the Institutional Animal Ethics Committee (IAEC) of IASST (IASST/IAEC/2016-17/002) and conducted following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

Dalton's Ascites Lymphoma Development in Mice
1 × 10 6 viable Dalton's ascites lymphoma (DLA) cells were injected into the mice through the intraperitoneal (I.P.) route. Tumour growth was confirmed by belly swelling and increased body weight, which was visible from 8-10 days post transplantation.

In Vivo Experimental Design
A total of 46 Swiss albino mice were selected for this study (n = 10), where for group-I n = 6. ART and 4-PB doses were scaled up according to the in vitro results. To develop DLA tumours, 0.2 mL of 1 × 10 6 DLA cells per mouse were inoculated. Drug treatments were started from the 8th day of DLA induction and continued for 8 days with 24 h interval.
Group-I: animals with no DLA tumour + 0.2 mL PBS (I.P). Group-II: animals with DLA tumour + 0.2 mL PBS (I.P). Group-III: animals with DLA tumour + 20 mg/kg ART (I.P). Group-IV: animals with DLA tumour + 50 mg/kg 4-PB (I.P). Group-V: animals with DLA tumour + 50 mg/kg 4-PB (I.P) + 20 mg/kg ART (I.P). Body weight changes were measured throughout the experimental period and cells from all treatment groups except Group-I were collected at the 16th day of the experiment to measure cell viability. At the 17th day, five animals from each group were sacrificed by decapitation and specimens of blood, liver and kidney were collected for biochemical and histopathological analysis. The other five animals were observed for a period of 50 days to estimate the mean survival time (MST) and percentage increase in lifespan (% ILS).

Histopathological Analysis
Pathological changes in liver and kidney were observed by histopathology analysis (Leica Biosystems, Nußloch, Germany). The livers and kidneys were collected in 10% buffered formaldehyde and kept for at least for 24 h. The tissues were dehydrated by gradient alcohol concentrations (50, 70, 90 & 100%) and xylene (1 h in each concentration). Further, the tissues were transferred to melted paraffin and kept for 1 h before embedding in paraffin blocks. Thin 5 µm sections were prepared using a microtome and the sections stained in hematoxylin and eosin. Stained sections were observed under a light microscope (10X) to observe pathological changes.

Cell Viability Assay
The trypan blue assay was conducted to determine cell viability and to observe cell cytotoxicity. Briefly, 0.1 mL of trypan blue (0.4 %) solution was mixed with 0.1 mL of DLA cells that were collected on day 17. The cells were quantified using an automated cell Five animals in each group (Except G-I) were observed for 50 days to measure mortality. A Kaplan-Meier curve was plotted to represent percent survival fraction using Prism, version 6.01 software (GraphPad Software Inc., San Diego, CA, USA).

Statistical Analysis
The degree of significance was determined using an unpaired, two-tailed t-test, and the IC50 dosage was determined using non-linear curve regression analysis. Experiments were carried out at least three times in duplicate and data points were represented as the mean ± SD (SD = standard deviation). One-way and two-way analysis of variance (ANOVA) and Tukey's post hoc test were applied for pairwise comparisons to determine the statistical significance of differences. Prism, version 6.01 (GraphPad Software Inc., San Diego, CA, USA), was used to analyze the data. Statistically significant values (p-value) for unpaired two-tailed t-test are provided in the Result section. For one-way and two-way analysis of variance (ANOVA) the p-values are * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

4-PB Cytotoxicity on MCF-7 and MDA-MB-231 Cells as a Consequence of HDAC Activity Inhibition
Initial MTT date assay suggested that the 4-PB significantly reduced the cell viability of MCF-7 and MDA-MB-231 cells at different concentrations when administered for 48 h ( Figure 1a). However, the effect of 4-PB on MCF-7 cells was more pronounced than on MDA-MB-231 cells. Using a 1 mM concentration of 4-PB, we observed 84% cell viability of MCF-7 cells, which further decreased to 68% at 5 mM concentration. We chose an intermediate dose of 5 mM of 4-PB to further assess the synergism of 4-PB with ART in MCF-7 cells.
As 4-PB-mediated growth inhibition was known to act by inhibiting HDAC [19], we further performed an HDAC activity assay by treating MCF-7 cells with 4-PB with or without ART ( Figure 1b). HDAC activity analysis using treated MCF-7 cell lysate showed a significant drop in the 4-PB (p = 0.0079) and 4-PB cells (p = 0.0082) treated with ART when compared with untreated control lysate fluorescence. No inhibition of HDAC was observed in ART-only treated cells.

4-PB Enhanced the Dose-Dependent Cytotoxicity of ART
To probe into the effect of ART on MCF-7 cells, a cell viability assay was performed. MCF-7 cells were treated with different doses of ART in the presence or absence of a constant dose of 4-PB (5-mM). Initially, ART treatment for 48 h showed a dose-dependent decrease in the cell viability of MCF-7 cells as depicted in Figure 1c. However, a significant (p = 0.0020) enhancement in the cell cytotoxicity of MCF-7 cells was observed when a similar treatment was performed in the presence of 4-PB. In particular, we observed that 55.56 ± 5.21 µM concentration of ART was required to reach its IC 50 , while for ART + 4-PB the IC 50 concentration was significantly reduced to only 24.71 ± 3.44 µM (Figure 1d). value of ART and ART in combination with 5 mM 4-PB was calculated using non-linear curve regression analysis and the level of significance were calculated using unpaired two tailed t-test (n = 3, p = 0.0020). Data are represented as mean ± SD (n = 4) in all the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

4-PB Enhanced the Dose-Dependent Cytotoxicity of ART
To probe into the effect of ART on MCF-7 cells, a cell viability assay was performed. MCF-7 cells were treated with different doses of ART in the presence or absence of a constant dose of 4-PB (5-mM). Initially, ART treatment for 48 h showed a dose-dependent decrease in the cell viability of MCF-7 cells as depicted in Figure 1c. However, a significant (p = 0.0020) enhancement in the cell cytotoxicity of MCF-7 cells was observed when a similar treatment was performed in the presence of 4-PB. In particular, we observed that 55.56 ± 5.21 µ M concentration of ART was required to reach its IC50, while for ART + 4-PB the IC50 concentration was significantly reduced to only 24.71 ± 3.44 µ M (Figure 1d).

Induction of Apoptosis by ART and/or 4-PB
Two independent sets of flow cytometry-based tests were carried out to establish that ART and/or 4-PB treated cells were undergoing apoptosis. First, PI dye was used to analyze the apoptotic nuclei, and then PE Annexin V and the 7-AAD test were used to distinguish between early, late, and necrotic apoptotic cells. and ART in combination with 5 mM 4-PB was calculated using non-linear curve regression analysis and the level of significance were calculated using unpaired two tailed t-test (n = 3, p = 0.0020). Data are represented as mean ± SD (n = 4) in all the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Induction of Apoptosis by ART and/or 4-PB
Two independent sets of flow cytometry-based tests were carried out to establish that ART and/or 4-PB treated cells were undergoing apoptosis. First, PI dye was used to analyze the apoptotic nuclei, and then PE Annexin V and the 7-AAD test were used to distinguish between early, late, and necrotic apoptotic cells.
Separation of the apoptotic cell population from the living cell population may be accomplished quickly, accurately, and repeatedly using PI staining. After 48 h of treatment, 4-PB showed an apoptotic cell population of 41.69%, and ART showed an apoptotic cell population of 65.12%. When we treated the MCF-7 cells with the drug combination, the extent of the apoptotic cell population increased drastically to 87.03% (Figure 2a).
We used the PE Annexin V and 7-AAD flow cytometric test to further confirm the results generated by the PI apoptotic assay (Figure 2b). The cells were stained with PE Annexin V and 7-AAD once the treatment period was over. Data from flow cytometry supported the observation that 4-PB increased ART's cytotoxic impact. We found 23.21% and 41.02% apoptotic cell populations (early and late apoptotic combined) after treatment with 4-PB and ART, respectively. When a combination of drugs was used, the effect was enhanced to 61.22% for the apoptotic cell population.
We used the PE Annexin V and 7-AAD flow cytometric test to further confirm the results generated by the PI apoptotic assay (Figure 2b). The cells were stained with PE Annexin V and 7-AAD once the treatment period was over. Data from flow cytometry supported the observation that 4-PB increased ART's cytotoxic impact. We found 23.21% and 41.02% apoptotic cell populations (early and late apoptotic combined) after treatment with 4-PB and ART, respectively. When a combination of drugs was used, the effect was enhanced to 61.22% for the apoptotic cell population.

Isobologram Depicted the Interaction of 4-PB with ART Is Synergistic
When MCF-7 cells were treated with 4-PB and ART, each of the drugs alone was able to induce apoptosis. However, when treated with the two drugs combined, the effect was substantially greater compared to that of the individual drugs. We investigated the nature of the interaction between these two drugs by plotting isobologram based on the Median-Effect Equation (Chou) and the Combination Index Theorem (Chou-Talalay). The combination index (CI) was used to express antagonism (CI > 1), additivity (CI = 1) or synergism (CI < 1). The CI was calculated using CompuSyn software.
For computing the nature of the interaction between 4-PB and ART, we designed an experiment keeping the concentration of 4-PB constant (i.e., 5-mM) while the concentration of ART was varied. Based on the available information about the mode of action of 4-PB and ART, we assumed that the 4-PB does not preclude the effect of ART and vice versa, thus they can be regarded as mutually non-exclusive. At the lowest concentration (4-PB + 10 µ M ART), 23% apoptosis was induced in MCF-7 cells, with a CI value of 1.3, indicating the interaction was close to additive. In contrast, when the concentration of ART was

Isobologram Depicted the Interaction of 4-PB with ART Is Synergistic
When MCF-7 cells were treated with 4-PB and ART, each of the drugs alone was able to induce apoptosis. However, when treated with the two drugs combined, the effect was substantially greater compared to that of the individual drugs. We investigated the nature of the interaction between these two drugs by plotting isobologram based on the Median-Effect Equation (Chou) and the Combination Index Theorem (Chou-Talalay). The combination index (CI) was used to express antagonism (CI > 1), additivity (CI = 1) or synergism (CI < 1). The CI was calculated using CompuSyn software.
For computing the nature of the interaction between 4-PB and ART, we designed an experiment keeping the concentration of 4-PB constant (i.e., 5-mM) while the concentration of ART was varied. Based on the available information about the mode of action of 4-PB and ART, we assumed that the 4-PB does not preclude the effect of ART and vice versa, thus they can be regarded as mutually non-exclusive. At the lowest concentration (4-PB + 10 µM ART), 23% apoptosis was induced in MCF-7 cells, with a CI value of 1.3, indicating the interaction was close to additive. In contrast, when the concentration of ART was increased (20, 50 100, and 250 µM) the CI value was decreased to 0.66, 0.77, 0.48, and 0.9 (indicating synergism) with an apoptosis of 49, 60, 85, and 89% of cells, respectively. We observed that when the concentration of 4-PB was kept constant and the concentration of ART was increased, the value of CI also decreased with up to a 250 µM concentration of ART, suggesting an increase in synergism between the two drugs (Figure 3a,b).

4-PB and ART Increased Oxidative Stress in MCF7 Cells
To investigate the mechanism of cell death induced by 4-PB and ART, we initially examined the role of these drugs in increasing oxidative stress in the cells. The level of intracellular ROS generated was estimated using intracellular peroxide-dependent oxidation of DCFH-DA to form fluorescent DCF. Fluorescence of DCF increases proportionally with the increase in the intracellular ROS generation. To measure ROS, the MCF-7 cells were treated with ART and/or 4-PB in the presence or absence of NAC for 8 h at 37 • C. An increase in DCF fluorescence was noticed in the treated samples with 4-PB or ART ( Figure 4a); however, the fluorescence intensity was considerably more in case of ART-treated samples, suggesting an extensive ROS generation. Consequently, the fluorescence intensity increased sharply in samples treated with the combination of both the drugs. The addition of NAC to the medium of the treated samples attenuated DCF fluorescence to that of control fluorescence, implying that the increase in fluorescence was due to ROS generation. increased (20, 50 100, and 250 µ M) the CI value was decreased to 0.66, 0.77, 0.48, and 0.9 (indicating synergism) with an apoptosis of 49, 60, 85, and 89% of cells, respectively. We observed that when the concentration of 4-PB was kept constant and the concentration of ART was increased, the value of CI also decreased with up to a 250 µ M concentration of ART, suggesting an increase in synergism between the two drugs (Figure 3a,b).

4-PB and ART Increased Oxidative Stress in MCF7 Cells
To investigate the mechanism of cell death induced by 4-PB and ART, we initially examined the role of these drugs in increasing oxidative stress in the cells. The level of intracellular ROS generated was estimated using intracellular peroxide-dependent oxidation of DCFH-DA to form fluorescent DCF. Fluorescence of DCF increases proportionally with the increase in the intracellular ROS generation. To measure ROS, the MCF-7 cells were treated with ART and/or 4-PB in the presence or absence of NAC for 8 h at 37 °C. An increase in DCF fluorescence was noticed in the treated samples with 4-PB or ART ( Figure  4a); however, the fluorescence intensity was considerably more in case of ART-treated samples, suggesting an extensive ROS generation. Consequently, the fluorescence intensity increased sharply in samples treated with the combination of both the drugs. The addition of NAC to the medium of the treated samples attenuated DCF fluorescence to that of control fluorescence, implying that the increase in fluorescence was due to ROS generation.

Cell Cycle Perturbation after Treatment with ART and/or 4-PB
Cytofluorimetric analysis of cells after synchronisation and treatment with ART and/or 4-PB showed that MCF-7 cells were progressively blocked in the G0/G1 phase of the cell cycle. However, after treatment with the drug combination, the percentage of the

Cell Cycle Perturbation after Treatment with ART and/or 4-PB
Cytofluorimetric analysis of cells after synchronisation and treatment with ART and/or 4-PB showed that MCF-7 cells were progressively blocked in the G0/G1 phase of the cell cycle. However, after treatment with the drug combination, the percentage of the cell population in the G0/G1 phase was considerably higher than in cells exposed to individual drugs alone. When treated with 4-PB and ART alone, the percentages of cells in the G0/G1 phase of the cell cycle were 69.38% and 71.82%, respectively. The G0/G1 phase population reached 83.84% after treatment with both 4-PB + ART combined. The data suggested that 4-PB enhanced the G0/G1 arrest induced by the ART treatment (Figure 4b).

Mechanism Underlying 4-PB Mediated Enhanced ART DNA Damaging Effect
We provided evidence that 4-PB enhances the effect of ART in MCF-7 cells, which led us to find the underlying mechanism behind the synergism showed by 4-PB and ART.
As an initial investigation into the underlying mechanism, we examined the effect of 4-PB on the expression of well-known DDR proteins, with or without the treatment involving ART.

Therapeutic Effectivity of Combinated Drusg in a Murine Model
For the assessment of the potentiality of the drug combination, the mice were split into five groups (n = 10). Except in group 1, DLA was induced in all other groups. It took 8 days for the tumour to attain an appropriate volume for drug treatment (swelling of the peritoneum was due to the accumulation of ascitic fluid). Administration of the drug was started after the tumour was formed (from the 8th day). On the 17th day, a cell viability assay was performed and the acquired data (Table 1) showed a significant decrease in the viable cell count after treatment with the combination drug compared with the other treatment groups (4-PB or ART). Along with cell viability, reduction in the volume of the tumour followed the same pattern as when 4-PB and ART were administered simultaneously in DLA mice, and the effectivity was greater compared with single drug treatments Figure 5. Expression analyses of DDR pathways. mRNA expression levels of DDR elements of the NHEJ pathway. Ku70, Ku80, XRCC4 and DNA-PKcs were assessed using qPCR (n = 3). Statistically significant values were taken as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Therapeutic Effectivity of Combinated Drusg in a Murine Model
For the assessment of the potentiality of the drug combination, the mice were split into five groups (n = 10). Except in group 1, DLA was induced in all other groups. It took 8 days for the tumour to attain an appropriate volume for drug treatment (swelling of the peritoneum was due to the accumulation of ascitic fluid). Administration of the drug was started after the tumour was formed (from the 8th day). On the 17th day, a cell viability assay was performed and the acquired data (Table 1) showed a significant decrease in the viable cell count after treatment with the combination drug compared with the other treatment groups (4-PB or ART). Along with cell viability, reduction in the volume of the tumour followed the same pattern as when 4-PB and ART were administered simultaneously in DLA mice, and the effectivity was greater compared with single drug treatments (Figure 6a). The above finding was also reflected in the lifespan of the mice (Figure 6b). In the control group, all the DLA mice died at the end of the 18th day after tumour inoculation, while the 4-PB or ART-treated mice were alive for up to 59 days but eventually died at the end of the observation period. However, the group receiving the combination treatment was alive till the end of the observation period. Although both drugs individually caused an effective reduction in cell viability as well as shrinking of tumour volume, both failed to increase the lifespan of the mice, while the combination drug did successfully increase the lifespan of the mice. DLA-bearing mice were observed to have elevated levels of WBC and reduced levels of Hb and RBC count. Treatment with the combination drugs reversed these conditions and restored the levels of WBC, RBC, and Hb counts to normal (Table S1). A significant decline in the liver function enzymes SGOT, SGPT & ALP was observed compared to the control group (Table S2). In Figure 6c, histopathological examination of liver and kidney tissues show a remarkable recovery in conditions that changed due to the DLA induction. For instance, in kidney, glomerular atrophy, tubular congestion, infiltration of cells and deformed epithelial cells were seen; however, the conditions reverted to normal with a decreased occurrence of tubular, glomerular and blood vessel congestion in the treated groups. In the liver, an abnormal arrangement of hepatocytes with polymorphic neutrophil infiltration, hepatic fibrillation, haemorrhage and perinuclear clumping of cytoplasm was observed in control group. Interestingly, in the treated group, reduction of damage of normal cellular ultrastructures comparable to normal animals was observed. Overall, these changes were more profound in the combination drug-treated group compared to the individual drug treatment regime.

Discussion
In this study, we showed that 4-PB enhanced the cytotoxicity of the traditional antiparasitic drug ART against a breast cancer cell line, as well as DLA, in mice. 4-PB DLA-bearing mice were observed to have elevated levels of WBC and reduced levels of Hb and RBC count. Treatment with the combination drugs reversed these conditions and restored the levels of WBC, RBC, and Hb counts to normal (Table S1). A significant decline in the liver function enzymes SGOT, SGPT & ALP was observed compared to the control group (Table S2). In Figure 6c, histopathological examination of liver and kidney tissues show a remarkable recovery in conditions that changed due to the DLA induction. For instance, in kidney, glomerular atrophy, tubular congestion, infiltration of cells and deformed epithelial cells were seen; however, the conditions reverted to normal with a decreased occurrence of tubular, glomerular and blood vessel congestion in the treated groups. In the liver, an abnormal arrangement of hepatocytes with polymorphic neutrophil infiltration, hepatic fibrillation, haemorrhage and perinuclear clumping of cytoplasm was observed in control group. Interestingly, in the treated group, reduction of damage of normal cellular ultrastructures comparable to normal animals was observed. Overall, these changes were more profound in the combination drug-treated group compared to the individual drug treatment regime.

Discussion
In this study, we showed that 4-PB enhanced the cytotoxicity of the traditional antiparasitic drug ART against a breast cancer cell line, as well as DLA, in mice. 4-PB synergistically ameliorated the effect of ART in a dose-dependent manner by contributing to G1 arrest of the cell cycle, increasing ROS production. Additionally, it could subjugate the DDR pathways in the MCF-7 cells, thus amplifying the DNA damage effect of ART.
ART is recognised as a remarkable new generation of antimalarial drugs with no noticeable side effects or obvious adverse reactions in humans [20]. Recent experimental evidence suggested that ART may provide a therapeutic alternative in rapid disseminating cancers, without inducing drug-based resistance [21]. Although, a few reports of cell lines and laboratory animal toxicity have raised concerns due to high doses and prolonged use of ART [13], these findings have not been reported to date in humans taking numerous doses of artemisinin. Keeping this in mind, we devised a novel combination therapy involving ART and 4-PB, in which, 4-PB reduced the effective cytotoxic ART concentration synergistically when administered to an MCF-7 cell line and in a murine model. 4-PB has been effectively used in patients with hyperammonemia and urea cycle disorders (UCDs) involving deficiencies of carbamyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), or argininosuccinic acid synthetase (AS) [22]. Although, two HDACi, namely vorinostat (suberoylanilide hydroxamic acid, Zolinza) and depsipeptide (romidepsin, Istodax), received approval from the FDA for the treatment of cutaneous T-cell lymphoma in 2006 and 2009 [23], respectively, 4-PB's potentiality as an anti-cancer drug is still under evaluation in various clinical trials [24].
Our observations indicated the enhancement of the cytotoxity of ART when combined with 4-PB. We further investigated the role of 4-PB in heightening the effect of ART by analysing the distribution of cells in several phases of the cell cycle. 4-PB has been known to cause a G1 arrest in cells by increasing the expression of p21 protein [25]. Thus, when synchronised MCF-7 cells were treated with a combination drug, they showed a substantial accumulation of cells in G1 phase. Basically, 4-PB amplified G1 arrest after treatment with ART. In a similar fashion, we also observed the enhancement in ROS generation inside the cells after the combination treatment.
Our data imply that 4-PB has strong synergism with ART in inducing a profound level of apoptosis in dose-dependent manner. Based on CI values, we deduced that the interaction between 4-PB and ART was synergistic rather than additive. To figure out the underlying pathway responsible for the enhanced effect, we checked for the expression levels of DDR elements after drug treatment. To date, there has been no report of downregulation of DDR elements after the treatment with 4-PB in MCF-7 cells. The only notable exception is a report that demonstrated that the role of 4-PB in the attenuation of the expression of Bcl-XL, DNA-PK, Caveolin-1, and VEGF was behind the increase in the sensitivity towards radiation-induced apoptosis in prostate cancer cells [26]. To elucidate the exact molecular mechanism by which 4-PB exerts a synergistic effect with ART, we analysed DNA repair processes, as it was reported earlier that ART induces DNA damage in cancer cells mediated by ROS [27,28]. Based on these reports, we investigated the expression levels of key proteins involved in the nonhomologous end joining pathway, namely Ku70, Ku86, XRCC4, and DNA-PKcs, which is crucial in DDR processes [29,30]. The qPCR analysis of the expression of these proteins showed downregulation at mRNA level after treatment with 4-PB. However, when treated with ART alone, the levels of these proteins increased significantly. Interestingly, the elevation nin the expression of these proteins diminished when 4-PB was introduced with ART in the medium, suggesting the involvement of attenuation of Ku70, Ku86, XRCC4, and DNA-PKcs by 4-PB in enhancing the effect of the combination drug.
To further examine the efficacy of our combination drug cocktail, we used a murine model. We used DLA mice for a preliminary investigation of the drug cytotoxicity for our in vivo studies. As DLA mimics most of the cancer types and is easy to maintain, it was used to provide fast and reliable information of the cytotoxic effects of the drug in vivo [31][32][33].
DLA-bearing Swiss albino mice were initially tested for tolerable doses of ART and/or 4-PB. Once done, we started the treatment of the DLA mice with ART and/or 4-PB as described in Materials and Methods section. We found a reduction in the volume of the tumour after treatment with ART and/or 4-PB, both visually and by weighing the mice. However, the reduction in tumour volume was more pronounced in mice treated with the combination drug. The survival probability data corroborated with the above findings. After an observation period of 40 days, mice treated with the combination drug survived over 40 days while others died within the observation period. The histological parameters of the treated mice were improved drastically compared with the control DLA mice.
In conclusion, we present a novel combination of drugs that are already in use for the treatment of other diseases, as a probable anti-cancer therapy. This drug combination holds immense potential for further testing in clinical trials. In this study, we showed that 4-PB synergistically enhanced the efficacy of ART and potentiated its cytotoxicity against MCF-7 cells. The underlying mechanism behind this revolves around the overall attenuation of the DDR elements inside the cell. The combination therapy showed its effectivity in DLA mice model. Overall, a comprehensive understanding of these findings requires further studies. However, the observations reported here emphasise the need for persistent improvement of strategies for making human tumour cells susceptible to cancer therapies that kill cancer cells by inducing DNA damage.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/sci4040041/s1, Table S1: Effect of drug treatment on hematological parameters. Table S2: Effect of drug treatment on serum biochemical enzymes. Figure S1: Reduction in the tumour volume was easily visualised in above image, taken after the respective drug treatment.  Data Availability Statement: The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request. All data generated or analysed during this study are included in this published article (and its Supplementary Information Files).