Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Apoptosis, Oxidative Stress, and Cell Cycle Analysis

The current study continues the evaluation of the anticancer potential of three de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides—MM129, MM130, and MM131—against human cancer cells of HeLa, HCT 116, PC-3, and BxPC-3 lines. The pro-apoptotic activity of the investigated sulfonamides was shown by observations of changes in the mitochondrial transmembrane potential of the tested cells, externalization of phosphatidylserine on the cellular membrane surface, and cell morphology in microscopic imaging. The computational studies have shown that MM129 exhibited the lowest binding energy values when docked against CDK enzymes. In addition, the highest stability was shown for complexes formed between MM129 and CDK5/8 enzymes. All examined compounds induced cell cycle arrest in the G0/G1 phase in the BxPC-3 and PC-3 cells and simultaneously caused the accumulation of cells in the S phase in the HCT 116 cells. In addition, the increase in the subG1 fraction was observed in PC-3 and HeLa cells. The application of a fluorescent H2DCFDA probe revealed the high pro-oxidative properties of the tested triazine derivatives, especially MM131. In conclusion, the obtained results suggest that MM129, MM130, and MM131 exhibited strong pro-apoptotic properties towards investigated cells, mainly against the HeLa and HCT 116 cell lines, and high pro-oxidative potential as well. Moreover, it is suggested that the anticancer activity of the tested compounds may be associated with their ability to inhibit CDK enzymes activities.


Introduction
Nowadays, cancer is among the leading causes of death worldwide. Therefore, searching for new effective strategies for cancer treatment is among the most challenging tasks in medicine [1][2][3][4]. The evaluation of the pro-apoptotic properties of novel potential antineoplastic agents, as well as their impact on cell cycle phase distribution, seems to be crucial in modern drug design. Apoptosis is a highly regulated process that plays a fundamental role in eliminating unwanted or unnecessary cells. Apoptosis dysregulation is connected with autoimmune disorders, neurological diseases, and many types of cancer. Because apoptosis evasion is a hallmark of all types of cancer, inducing apoptosis in cells to limit their uncontrolled proliferation appears to be a feasible and highly successful strategy to combat many types of cancer [5,6]. CDK2, AKT serine/threonine-protein kinase, PD-1/PD-L1, Bruton's tyrosine kinase (BTK), phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) [59,60]. Furthermore, some studies suggest that the MM129 may be a potentially safe and welltolerated chemotherapeutic agent in colon cancer treatment [61]. Interestingly, MM129 is characterized by a similar chemical structure compared to seliciclib (roscovitine). This antineoplastic agent is the first selective oral CDK inhibitor that entered clinical trials [62]. Currently, several novel pyrazolo [4,3-e]tetrazolo [4,5-b] [1,2,4]triazine derivatives are at different stages of the in vitro and in vivo investigations. In this paper, we present the anticancer properties of MM129, MM130, and MM131 belonging to this group of structurally related compounds ( Figure 1). Recent studies have shown that MM129 may inhibit CDK2, AKT serine/threonine-protein kinase, PD-1/PD-L1, Bruton's tyrosine kinase (BTK), phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) [59,60]. Furthermore, some studies suggest that the MM129 may be a potentially safe and welltolerated chemotherapeutic agent in colon cancer treatment [61]. Interestingly, MM129 is characterized by a similar chemical structure compared to seliciclib (roscovitine). This antineoplastic agent is the first selective oral CDK inhibitor that entered clinical trials [62]. Our previous paper described the synthesis of MM129, MM130, and MM131 compounds. Furthermore, the high cyto-and genotoxic properties of the investigated triazine derivatives against the HeLa, HCT 116, PC-3, and BxPC-3 cancer cell lines have been demonstrated. Importantly, the investigated compounds were noticeably less cytotoxic toward normal cells-human foreskin fibroblast (Hs27) and human peripheral blood mononuclear cells (PBMCs). The IC50 values obtained in the MTT assay were shown in the Supplementary Material (Table S1) [63]. Therefore, we decided to investigate the pro-oxidative and pro-apoptotic properties of three novel derivatives of pyrazolo-triazine sulfonamides MM129, MM130, MM131 against four adherent human cancer cell lines-HeLa, HCT 116, PC-3, and BxPC-3. The assessment of the effect of these compounds on the cell cycle phase distribution was also evaluated. Moreover, we conducted computational studies (molecular docking and molecular dynamics techniques) to confirm our results from the cell cycle analysis with the potential ability of examined sulfonamides to inhibit CDKs. Our previous paper described the synthesis of MM129, MM130, and MM131 compounds. Furthermore, the high cyto-and genotoxic properties of the investigated triazine derivatives against the HeLa, HCT 116, PC-3, and BxPC-3 cancer cell lines have been demonstrated. Importantly, the investigated compounds were noticeably less cytotoxic toward normal cells-human foreskin fibroblast (Hs27) and human peripheral blood mononuclear cells (PBMCs). The IC 50 values obtained in the MTT assay were shown in the Supplementary Material (Table S1) [63]. Therefore, we decided to investigate the pro-oxidative and pro-apoptotic properties of three novel derivatives of pyrazolo-triazine sulfonamides MM129, MM130, MM131 against four adherent human cancer cell lines-HeLa, HCT 116, PC-3, and BxPC-3. The assessment of the effect of these compounds on the cell cycle phase distribution was also evaluated. Moreover, we conducted computational studies (molecular docking and molecular dynamics techniques) to confirm our results from the cell cycle analysis with the potential ability of examined sulfonamides to inhibit CDKs.  Among the examined cell lines, Hela cells showed the highest sensitivity for the proapoptotic activity of MM compounds, even after the 24 h incubation, where the apoptosis  induction for the IC 50 and 2×IC 50 compounds was significantly higher compared to the untreated cells (6.73% for the control group vs. 15.5-73.47% for experimental samples). Moreover, an increase in the percentage of apoptotic HCT 116 cells after their 24 h exposure to sulfonamides at the IC 50 and 2×IC 50 was noticed (6.92% for the untreated samples vs. 14.33-20.67% for experimental groups). Figure 2 shows the apoptosis induction in cancer cells following their 24 h, 48 h, and 72 h incubation with MM129, MM130, and MM131 compounds. Studied pyrazole-triazine derivatives induced time and concentration-dependent increase in the fraction of apoptotic cells. Among the examined cell lines, Hela cells showed the highest sensitivity for the pro-apoptotic activity of MM compounds, even after the 24 h incubation, where the apoptosis induction for the IC50 and 2×IC50 compounds was significantly higher compared to the untreated cells (6.73% for the control group vs. 15.5-73.47% for experimental samples). Moreover, an increase in the percentage of apoptotic HCT 116 cells after their 24 h exposure to sulfonamides at the IC50 and 2×IC50 was noticed (6.92% for the untreated samples vs. 14.33-20.67% for experimental groups).  Among the investigated compounds, MM129 exhibited the highest pro-apoptotic activity. MM129 at 1 2 IC 50 was the only one that induced apoptosis in HeLa cells following the 24 h of incubation. In addition, it was also the most effective toward PC-3 cells, where significant changes in the apoptotic cell fraction after the 48 h of exposure were observed even at the IC 50 .

Flow Cytometry Assessment of Annexin V Binding
The representative flow cytometry dot-plots for all cancer cell lines were demonstrated in Supplementary Materials (Figures S1-S12). The fraction of necrotic cells in all experimental series has not exceeded 15%, even after 72 h exposure of cells to the tested compounds in their highest concentrations (2×IC 50 ). Among the investigated compounds, MM129 exhibited the highest pro-apoptotic activity. MM129 at ½ IC50 was the only one that induced apoptosis in HeLa cells following the 24 h of incubation. In addition, it was also the most effective toward PC-3 cells, where significant changes in the apoptotic cell fraction after the 48 h of exposure were observed even at the IC50.

Dual
The representative flow cytometry dot-plots for all cancer cell lines were demonstrated in Supplementary Materials (Figures S1-S12). The fraction of necrotic cells in all experimental series has not exceeded 15%, even after 72 h exposure of cells to the tested compounds in their highest concentrations (2×IC50).   The results obtained in OA/EB staining confirmed the presence of morphological changes characteristic of apoptotic cells, such as shrinking and condensation of chromatin after exposure to the tested sulfonamides. Figure 4 shows the exemplary images from this experiment. The results obtained in OA/EB staining confirmed the presence of morphological changes characteristic of apoptotic cells, such as shrinking and condensation of chromatin after exposure to the tested sulfonamides. Figure 4 shows the exemplary images from this experiment.     After 48 h of exposure to the studied sulfonamides at their highest applied concentration (2×IC 50 ), significant alterations in ∆Ψm were observed in all tested types of cancer cells. Following the 24 h of incubation time, a significant disturbance of ∆Ψm was noticed only in Hela and HCT 116 cells. The most profound decrease in the ∆Ψm was observed in the HeLa cell line after 48 h exposure to MM129 (67.53 ± 7.94% of control value). Figure 6 presents the changes in ROS level observed after 2 h exposure of the tested cells to MM compounds. Examined sulfonamides induced concentration-dependent increase in ROS level in all tested cancer cell types. All studied compounds caused significant changes in HCT 116 and BxPC-3 cancer cells when compared to the negative control, even at the lowest concentrations applied. The same results were obtained for HeLa and PC-3 cells except for MM129, which significantly increased intracellular ROS level only in its higher concentrations (IC 50 and 2×IC 50 for the Hela cell line and 2×IC 50 for the PC-3 cell line). The administration of MM131 resulted in the most significant elevation of reactive oxygen species (ROS) levels across all the tested cancer cell lines, with an observed increase ranging from 206.3% to 316.3% compared to control conditions.   50 , and 2×IC 50 (obtained in the MTT assay) using MitoTracker Red CMXRos. Data are presented as the mean ± SD relative to the control group normalized to 100%. * p < 0.05 indicates a statistically significant difference compared to the negative control.

Determination of Intracellular ROS Level Using H 2 DCFDA
PC-3 cells except for MM129, which significantly increased intracellular ROS level only in its higher concentrations (IC50 and 2×IC50 for the Hela cell line and 2×IC50 for the PC-3 cell line). The administration of MM131 resulted in the most significant elevation of reactive oxygen species (ROS) levels across all the tested cancer cell lines, with an observed increase ranging from 206.3% to 316.3% compared to control conditions. . ROS level is expressed as the mean percentage of fluorescence intensity of H2DCFDA relative to control ± SD. Significant differences (p < 0.05) compared with negative control (*). . ROS level is expressed as the mean percentage of fluorescence intensity of H 2 DCFDA relative to control ± SD. Significant differences (p < 0.05) compared with negative control (*). h exposure to MM compounds. Of the sulfonamides tested, only MM129 at the IC50 concentration significantly affected cell cycle progression in HeLa cells. The incubation of the tested cells with MM129 caused an increase in the percentage (%) of cells in the subG1 phase, and simultaneous decrease in the number of cells (%) in the G0/G1 phase. In the case of HCT 116 cells, an accumulation of cells in the S phase and a reduction in the proportion of cells in the G2/M phase after treatment with triazine derivatives at the IC50 was observed. The cell cycle distribution of PC-3 cells showed that all tested MM compounds increased the number of cells (%) in the G0/G1 and subG1 phases and caused a decrease in the % of cells in the S phase. In contrast, an increase in the number of cells in the G0/G1 phase and a reduction in cells in the G2/M phase was observed following the exposure of BxPC-3 cells to examined sulfonamides at the IC50. The representative flow cytometry histograms for the tested cell lines were demonstrated in Supplementary Materials ( Figure  S13).

Molecular Docking
MM compounds/CDK complexes were analyzed according to their binding energy (kcal/mol) obtained during the molecular docking procedure as presented in Table 1.

Molecular Docking
MM compounds/CDK complexes were analyzed according to their binding energy (kcal/mol) obtained during the molecular docking procedure as presented in Table 1. MM129 exhibited the lowest binding energy values when docked against CDK enzymes. Therefore, it showed the best binding potential among the MM compounds (except for the docking of MM131 with CDK6). MM129 showed more profound binding potential compared with reference ligands of CDK enzymes (except for CDK9). The highest binding affinities were observed for CDK4 and CKD7 (binding energy of −9.48 and −9.84, respectively). However, we have decided to assess the molecular stability of MM129 with all the investigated CDK enzymes.

Molecular Dynamics Simulations
Sufficient stability of the MM129/CKDs complexes was observed during molecular dynamics simulation. The highest stability based on the observed parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), protein-ligand contacts, and conserved secondary structure were shown for complexes formed between MM129 and CDK5/8 enzymes. Thus, it has been supposed that MM129 executes the anticancer effect via synergistic targeting of both CDK5 and CDK8 enzymes.
The target enzyme CDK5 has 276 residues distributed in a macromolecular chain consisting of 2225 heavy atoms out of a total of 4505 atoms. The macromolecular target has 35% of secondary structures in the form of 27% of alpha helices and 8% of beta-strands, which were found to be conserved during the simulation process. MM129 possesses 32 heavy atoms out of a total of 51 atoms. Dynamic simulation of the macromolecular complex of MM129 against the CDK5 target has clearly shown that the RMSD for the fluctuation of the protein backbone was in-between 1.2-2.0 Å, which is well within the acceptable range. Similarly, the ligand MM129 showed some initial adjustment up to 5 ns within the active site to achieve the stable conformation followed by its stabilized vibrations within the range of 3.6-4.8 Å. Afterward, the complexed ligand attained the most stable conformation and remained stable throughout the simulation process. The RMSF of the macromolecular backbone was found to be well within the range of 0.4-2.0 Å throughout the simulation process. Macromolecular residues such as Ile10, Tyr15, Gly16, Val18, Cys83, Asp86, Lys128, Gln130, Asn131, Leu133, and Asn144 of CDK5 were found to interact with the ligand MM129 throughout the simulation process.
The target enzyme CDK8 has 346 residues distributed in a macromolecular chain consisting of 2855 heavy atoms out of a total of 5716 atoms. The macromolecular target has 35% of secondary structures in the form of 25% of alpha helices and 10% of beta-strands, which were found to be conserved during the simulation process. Dynamic simulation of the macromolecular complex of MM129 against the CDK8 target clearly showed that the RMSD for the fluctuation of the protein backbone was in-between 2.5-4.0 Å, which is well within the acceptable range. Similarly, the ligand MM129 showed stable conformation within the range of 3.0-4.2 Å and remained stable throughout the simulation process. The RMSF of the macromolecular backbone was found to be well within the range of 0.6-2.4 Å except for some terminal residues throughout the simulation process. Macromolecular residues, such as Val27, Arg29, Thr31, Lys52, Phe97, Asp98, Ala100, Asp103, Trp105, Lys153, Ala155, Leu158, and Asp173 of CDK8 were found to interact with the ligand MM129 throughout the simulation process. The RMSD of the macromolecular complex of MM129 against CDK5 and CDK8 observed during the 100 ns of the MD simulation is displayed in Figure 8. Trp105, Lys153, Ala155, Leu158, and Asp173 of CDK8 were found to interact with the ligand MM129 throughout the simulation process. The RMSD of the macromolecular complex of MM129 against CDK5 and CDK8 observed during the 100 ns of the MD simulation is displayed in Figure 8.

Discussion
In the current study, we have demonstrated the pro-apoptotic and pro-oxidative activity of MM129, MM130, and MM131 towards Hela, HCT 116, PC-3, and BxPC-3 cancer cells. Furthermore, our research included computational studies and analysis of cell cycle distribution in the tested cells after 24 h exposure to MM129, MM130, and MM131.
The obtained results of the Annexin V binding assay showed the pro-apoptotic activity of examined sulfonamides towards the HeLa, HCT 116, PC-3, and BxPC-3 cancer cell lines (Figure 2). Only the HeLa and HCT 116 cell lines showed significant changes in the apoptosis prevalence following 24 h exposure to the investigated compounds. A high number of apoptotic cells was detected following the treatment of the HeLa cells with MM129 and MM130 (73.47 ± 3.48% and 55.63 ± 1.27% of apoptotic cells at 2×IC50, respectively. Following 48 h incubation, all MM compounds demonstrated an enhancement in apoptotic rates across all the cancer cell lines when administered at their highest concentration (2×IC50). However, the most noticeable effect was observed for the HCT 116 and HeLa cells. The highest apoptotic response to the investigated triazine derivatives was observed after 72 h incubation of the tested cancer cell lines. The high apoptotic properties

Discussion
In the current study, we have demonstrated the pro-apoptotic and pro-oxidative activity of MM129, MM130, and MM131 towards Hela, HCT 116, PC-3, and BxPC-3 cancer cells. Furthermore, our research included computational studies and analysis of cell cycle distribution in the tested cells after 24 h exposure to MM129, MM130, and MM131.
The obtained results of the Annexin V binding assay showed the pro-apoptotic activity of examined sulfonamides towards the HeLa, HCT 116, PC-3, and BxPC-3 cancer cell lines (Figure 2). Only the HeLa and HCT 116 cell lines showed significant changes in the apoptosis prevalence following 24 h exposure to the investigated compounds. A high number of apoptotic cells was detected following the treatment of the HeLa cells with MM129 and MM130 (73.47 ± 3.48% and 55.63 ± 1.27% of apoptotic cells at 2×IC 50 , respectively. Following 48 h incubation, all MM compounds demonstrated an enhancement in apoptotic rates across all the cancer cell lines when administered at their highest concentration (2×IC 50 ). However, the most noticeable effect was observed for the HCT 116 and HeLa cells. The highest apoptotic response to the investigated triazine derivatives was observed after 72 h incubation of the tested cancer cell lines. The high apoptotic properties of MM129 and MM131 towards other cancer cells (HT-29 and DLD-1) were confirmed by other researchers [60,64].
During microscopic analysis typical morphological features of apoptosis, including chromatin condensation, cell shrinkage, nuclear fragmentation, and membrane blebbing, were noticed following the treatment of cells with MM compounds (Figure 4). Furthermore, a quantitative analysis of the AO/EB double-stained cells confirmed the pro-apoptotic potential of examined sulfonamides observed in the Annexin V binding assay (Figure 3). A significant increase in the number of apoptotic cells in all the cancer cell lines was noticed after 48 h incubation with investigated compounds at their highest concentration. Similarly to the Annexin V assay, the results of AO/EB revealed that HeLa was the most sensitive cell line to the pro-apoptotic activity of the compounds. In addition, the results of the AO/EB dual staining confirmed the pro-apoptotic activity of MM131 towards DLD-1 and HT-29 cancer cells [60]. Furthermore, a similar pattern was observed in the case of other triazine derivatives against several cancer cell lines.
In the present work, all studied sulfonamides depleted ∆Ψm in all the evaluated cell lines following 48 h of treatment. Additionally, a decrease in ∆Ψm was observed in the HeLa and HCT 116 cells after 24 h exposure to MM compounds in their highest concentration ( Figure 5). The results of Hermanowicz et al. [64] also showed the ability of MM129 to decrease the ∆Ψm in the DLD-1 and HT-29 cells. Moreover, the literature data have shown a similar activity of other MM compounds towards the different cancer cell lines [65,66]. According to the study of Ly et al. [67], loss of ∆Ψm may be an early event of the apoptotic-signaling pathway. However, ∆Ψm reduction may also be a consequence of apoptosis when it is triggered by the opening of the mitochondrial permeability transition pore (PTP) [67]. As mentioned above, the studies have indicated that the reduction of ∆Ψm is the effect of the opening of PTP. In contrast, other research works have suggested the opposite process-the loss of ∆Ψm is responsible for the opening of PTP [68]. Undoubtedly, loss of the ∆Ψm and opening of the PTP are associated with the release of pro-apoptotic proteins, such as cytochrome c, apoptosis-inducing factor (AIF), and procaspase-9. As a consequence, this leads to a circle of self-perpetuating apoptotic changes [67,69,70].
Moreover, the assessment of the pro-oxidative properties of examined compounds was conducted. All the investigated sulfonamides induced a concentration-dependent increase in ROS formation in all investigated cancer cell types following 2 h exposure ( Figure 6). ROS accumulation may cause DNA strand break formation, mitochondrial DNA lesions, and degradation of mitochondrial DNA [16]. The high pro-oxidative activity of tested triazine derivatives is consistent with the data obtained from our previous studies, where examined compounds exhibited high genotoxicity against the same cancer cell lines. Similarly to the genotoxicity studies, MM131 showed the most potent pro-oxidative potential among the tested compounds [63]. Furthermore, research has shown that the accumulation of ROS-related damages in lipids, proteins, and DNA may be responsible for the suppression of tumor cell proliferation and growth via cell cycle arrest and apoptosis. [71,72]. Literature data have suggested that raised cytosolic calcium ion levels and increased formation of ROS cause a decrease in electrolyte transport across the mitochondrial membrane, leading to the opening of PTP and collapse of ∆Ψm [68,[73][74][75]. As a consequence, various pro-apoptotic molecules are released, activating the mitochondrial apoptosis pathway [15,73,[76][77][78][79][80][81][82][83][84][85][86]. In addition, ROS can also enhance the death receptor-mediated apoptosis pathway by downregulation the FLICE-inhibitory protein (c-FLIP) half-life [87]. Moreover, ROS may bind to death receptors containing the death domain (DD), and thus initiate the extrinsic pathway of apoptosis through interaction with other adaptor proteins with DDs [88,89]. In addition, the activation of apoptosis by ROS is the result of mitochondrial damage and activation of poly(ADP-ribose) polymerase (PARP) and apoptosis signal-regulating kinase 1 (ASK1) [90]. Therefore, we conclude that the ROS formation observed in the tested cancer cells after the incubation with examined compounds may be the cause for the further loss of ∆Ψm.  (Figure 7). Interestingly, the varied activity of the same antineoplastic drug against a diversity of cancer cell lines is consistent with earlier research findings. As an example, 5-fluorouracil (5-FU) increased the accumulation of cells in the G0/G1 phase in gastric cancer cells AGS and some colorectal cancer (CRC) cell lines, such as HT-29 and DLD-1 while also arresting oral cancer cells HSC-4 and colon cancer cells SW620 in the S phase [60,[91][92][93]. Our previous research has shown that all examined triazine derivatives are highly genotoxic toward the tested cell lines [63]. Interestingly, the most noticeable increase in the number of cells in the S phase in HCT 116 cells was observed in the case of MM131. This is consistent with our previous results, where MM131 induced the highest levels of DNA damage among the tested sulfonamides [63]. In turn, the elongated G0/G1 phase also provides the cell more time to repair DNA damage, preventing the it from entering the S phase. The genotoxic study of the investigated triazine derivatives has presented their high genotoxic activity not only against HCT 116 but also towards PC-3 and BxPC-3 cells [63]. Thus, the cell cycle arrest in the G0/G1 phase in PC-3 and BxPC-3 cells after the incubation with MM compounds may also be the effect of the accumulation of DNA damage.

The cell cycle distribution of PC-3 cells showed a significant increase in the proportion of the cells in the G0/G1 phase and a reduction in the number of cells (%) in the S phase
Finally, our results of molecular docking have shown the high potential of MM129, as well as MM130, and MM131 to bind with CDK enzymes. MM129 exhibited the lowest binding energy values among examined sulfonamides, especially for CDK4 and CDK7 ( Table 1). The results of molecular dynamics simulations confirmed sufficient stability of the MM129/CKDs complexes. The highest stability was observed for the associations of MM129 with CDK5 and CDK8 enzymes. In the study of Hermanowicz et al. [59], an upregulation of TP53 and simultaneous downregulation of CDK2 was observed after the exposure of the HT-29 and DLD-1 cell lines to MM129. The cell cycle arrest in the G0/G1 phase was noticed in both CRC lines after the incubation with MM129. Thus, the change that was seen in those cells was most likely induced by an increase in TP53 expression in conjunction with a downregulation of CDK2 activity [59]. Therefore, we conclude that the interaction of investigated compounds with CDKs may be among the potential causes of observed disturbances in the cell cycle distribution in the tested cells. Cancer cells were maintained in a humidified incubator at 37 • C in a 5% CO 2 atmosphere and were systematically scanned for mycoplasma contamination. The proper number of cultivated cells was managed by their regular passaging at 90% confluence twice weekly using 0.025% trypsin/EDTA. Additionally, to maintain stable growth conditions for cell culture, the medium was replaced every 48 h. Each experiment was performed on cells from three independent passages and included a negative control (cells treated with DMSO at <0.5%). The concentrations for all experiments were based on the values obtained in the MTT assay after 72 h of cell incubation with examined compounds ( 1 2 IC 50 , IC 50 , and 2×IC 50 ) [63].

Flow Cytometry Assessment of Annexin V Binding
Evaluation of the induction of apoptosis in the HeLa, HCT 116, PC-3, and BxPC-3 cell lines was performed using the FITC Annexin V Apoptosis Detection Kit I via flow cytometry analysis. The translocation of the PS on the outer side of the cell membrane was associated with PCD. Annexin V conjugated with fluorescein isothiocyanate (FITC) binds to PS, which allows the identification of apoptotic cells, while not membrane-permeable PI binds to the DNA of necrotic cells. Double staining identifies independently early and late apoptosis (cells stained only with FITC or with both dyes, respectively) [94].
Cancer cells were cultured for 24 h in controlled conditions (5% CO 2 ; 37 • C) on 6-well plates, and then exposed to MM compounds in different concentrations ( 1 2 IC 50 , IC 50 , and 2×IC 50 ) for 24 h, 48 h, or 72 h. After incubation, the cells were trypsinized and transferred to conical tubes, then washed twice with cold PBS. Following centrifuging (10 min, 4 • C, 1400 rpm), the cells were suspended in Annexin V-binding buffer and stained with PI and FITC-labelled Annexin V for 15 min at room temperature in the dark. Additional controls were used to set up compensation and quadrants were included (unstained cells and cells dyed only with FITC Annexin V or PI). Measurements were performed using a flow cytometer (LSR II, Becton Dickinson, San Jose, CA, USA) at the wavelength of 530 nm. The obtained data (10,000 events per sample) were analyzed in FlowJo 10.8.1 software.

Dual Acridine Orange/Ethidium Bromide (AO/EB) Fluorescent Staining
Acridine orange (AO) intercalates into the DNA of living cells after penetration of their cell membrane and accumulates in lysosomes, emitting green and red fluorescence, respectively. In contrast, ethidium bromide (EB) interacts only with the DNA and RNA of membrane-damaged cells, emitting red fluorescence. This method allows the observation of the development of apoptotic bodies and detects nuclear alterations, which are both associated with apoptosis. The morphological changes in chromatin condensation in the dyed nucleus, combined with different uptake of AO and EB, allows for distinguishing three categories of cells as follows: viable (green-stained nucleus uniformly distributed in the center of the cell), apoptotic (orange-stained cell nuclei with condensation and chromatin clumping; the presence of cell membrane blebbing indicating the apoptotic bodies formation) and necrotic (increased cell's volume; uneven orange-red fluorescence without a sharp outline; cell near disintegration) [95,96].
The cells were seeded for 24 h in controlled conditions (5% CO 2 ; 37 • C) on 12-well plates, and then were incubated with MM compounds in different concentrations ( 1 2 IC 50 , IC 50 , and 2×IC 50 ) for 48 h. After being cultured, the cells were trypsinized, transferred to conical tubes, and then centrifuged (10 min, 4 • C, 1400 rpm). Afterward, the supernatant was removed, and the precipitate was diluted in 25 µL of PBS and transferred to the Eppendorf tube. The suspension was mixed with dual fluorescent staining solution (1 µL) containing 100 µg/mL AO and 100 µg/mL EB, transferred to glass slides, and then covered with a coverslip. The morphology of the cells was conducted, and 200 cells were counted within 10 min using a fluorescent microscope (Olympus BX60 F5, Olympus Optical Co., Ltd., Nagano, Japan) at 360 nm.

Mitochondrial Membrane Potential (∆Ψm)
Disruption of mitochondrial membrane potential is among the earliest changes associated with the induction of apoptosis [10]. As a result of the increased permeability of the internal and external mitochondrial membrane, the mitochondrial proteins, such as apoptosis-inducing factor (AIF) and cytochrome c migrate into the cytosol leading to the activation of the intrinsic apoptosis pathway. MitoTracker CMXRos, which contains thiol-reactive chloromethyl moiety passively diffuses across the plasma membrane. Accumulation of fluorescence probe in active mitochondria allows the measurement of changes in the ∆Ψm of the tested cells [67,97] Cancer cells were seeded for 24 h in controlled conditions (5% CO 2 ; 37 • C) on 96-well plates and then were treated with investigated compounds in different concentrations ( 1 2 IC 50 , IC 50 , and 2×IC 50 ) for 24 h or 48 h. Following incubation, the cells were washed with PBS and stained with the probe at the final concentration of 0.1 µM for 40 min at 37 • C in the dark. The transmembrane mitochondrial potential was measured using a microplate reader at the wavelength of 579/599 nm.

Determination of Intracellular ROS Level by H 2 DCFDA
ROS are associated with the regulation of apoptosis pathways mediated via endoplasmic reticulum (ER), death receptors, and mitochondria [69]. Accordingly to the protocol described previously by Ruiz-Leal and George [98], H 2 DCFDA was used to determine the level of intracellular ROS formation. H 2 DCFDA is a cell-permeable stain widely used for the quick and effective detection of total intracellular oxidants. The probe can diffuse across an intact cellular membrane and then is hydrolyzed to 2 ,7 -dichlorodihydrofluorescein (H 2 DCF) by intracellular esterases active in living cells. Finally, ROS oxidate dichlorodihydrofluorescein to a highly fluorescent 2 ,7 -dichlorofluorescein (DCF).
Cancer cells were cultured for 24 h in controlled conditions (5% CO 2 ; 37 • C) on black 96-well plates. Afterward, the culture medium was replaced by H 2 DCFDA dissolved in PBS (final concentration was 20 mM), and the cells were incubated for 20 min in the dark. Following incubation, cell lines were exposed to examined sulfonamides in different concentrations ( 1 2 IC 50 , IC 50 , and 2×IC 50 ). After 120 min of incubation, the fluorescence was measured using a microplate reader at the wavelength of 485/535 nm using the SpectraMax ® i3xMulti-Mode Detection Platform.

Cell Cycle
The effect of MM compounds on the cell cycle was determined by flow cytometry. The cell cycle distribution of a sample was estimated by staining the DNA with a fluorescent dye, such as PI, which binds to the DNA stoichiometrically. The analysis of the cellular DNA content frequency histograms reveals the differentiation of cells in the G0/G1, S, and G2/M phases. Furthermore, this approach allows the identification of aneuploid populations, as well as apoptotic cells with fractional DNA content (subG1) [99].
The cells were seeded for 24 h in controlled conditions (5% CO 2 ; 37 • C) on 6-well plates, and then were incubated with MM compounds at 1 2 IC 50 and IC 50 for 24 h. After incubation, the cells were trypsinized and transferred to conical tubes, then washed with PBS. Following centrifuging (10 min, 4 • C, 1400 rpm), the cells were fixed with cold ethanol (70%), then washed with PBS. Afterward, the cells were treated with 10 ug/mL of RNase A solution and stained with 5 µg/mL of PI for 30 min at 37 • C in the dark. The measurements were performed using a flow cytometer (LSR II, Becton Dickinson) at the wavelength of 530 nm. The obtained data (10,000 events per sample) were analyzed in FlowJo 10.8.1 software.

Molecular Docking
In an attempt to explore the potential inhibitory effects of MM129, MM130, and MM131 on CDK enzymes, we have performed molecular docking. MM compounds were sketched in ChemDraw 8.0 software and their three-dimensional structure was obtained following energy minimization using an MM2 force field. To get the ligands ready for molecular docking simulation, we used AutoDock Tools (The Scripps Research Institute, La Jolla, CA, USA) to find aromatic carbons and rotatable bonds, configure the automated torsion number, margin non-polar hydrogens, and add Gasteiger charges.
The grid box values were adjusted following the docking of reference ligands already complexed in the PDB structure after throughout observation of the drug's conformational poses. The grid parameters for each CDK enzyme were saved in a grid parameter file (GPF). The Autogrid utility from the Autodock suite was used to create the additional map files required for running the molecular docking simulations ( Table 2).

Molecular Dynamics Simulations
To validate the obtained docking outcome for the thermodynamic stability of the macromolecular drug-receptor complex concerning time, a molecular dynamics (MD) simulation for 100 ns for the macromolecular complex of ligand MM129 with all the CDKs considered in the current research paradigm was executed. MD simulation was performed at the molecular level at a constant temperature of 300 K and constant pressure conditions. Schrödinger-Desmond (version 2019.4) module of Maestro software was used to perform dynamics simulation by using OPLS3e force field and solvating it in an explicit water box of size 10. The single point charge (SPC) model was used to infer the water molecules [100]. The usage of the OPLS3e force field and the SPC water model for macromolecular complexes with small ligands were previously described to yield the optimum reproducible results [101]. The complex was neutralized by adding the contrary-charged ions followed by their energy minimization [102]. The long-range electrostatic connections between the macromolecule and the complexed ligand were computed by applying the particle-mesh Ewald (PME) method with 0.8 grid spacing and a cutoff radius of 9.0 for Coulomb interactions after the NPT ensemble, MD simulation was run.

Statistical Analysis
All data are presented as the mean values with standard deviations (SD) obtained from three independent experiments. The statistical analysis was evaluated with GraphPad Prism 8.0 software system (GraphPad Prism Software Inc., San Diego, CA, USA), by using one-way ANOVA with Tukey's test (post hoc multiple comparisons in distributions notdeparting from normality). A p-value of <0.05 was considered significant.

Conclusions
The presented research demonstrates the diverse, cell-line-dependent pro-oxidative and pro-apoptotic activity of novel pyrazolo [4,3-e]tetrazolo [1,5-b] [1,2,4]triazine sulfonamides, such as MM129, MM130, and MM131, on HeLa, PC-3, HCT 116, and BxPC-3 cells. The results from the H 2 DCFDA and apoptotic assays (Annexin V binding assay, MitoTracker assay, and AO/EB) proved the high pro-oxidative and pro-apoptotic activity of MM129, MM130, and MM131 in vitro. All investigated compounds also affected the cell cycle distribution inducing the cell-line-dependent cycle arrest in the G0/G1 or S phase. Our previous data have shown the high genotoxic potential of examined compounds toward tested cancer cells [63]. Therefore, we consider that the increase in apoptosis and the changes in the cell cycle distribution result from the strong genotoxicity of MM129, MM130, and MM131. Furthermore, based on computational studies, we conclude that the ability of the investigated sulfonamides to change the distribution of the cell cycle may result from their potential for CDKs inhibition. Moreover, we suggest that a high level of ROS may enhance the genotoxic and pro-apoptotic properties of the investigated triazine derivatives (Figure 9). tween the macromolecule and the complexed ligand were computed by applying the particle-mesh Ewald (PME) method with 0.8 grid spacing and a cutoff radius of 9.0 for Coulomb interactions after the NPT ensemble, MD simulation was run.

Statistical Analysis
All data are presented as the mean values with standard deviations (SD) obtained from three independent experiments. The statistical analysis was evaluated with GraphPad Prism 8.0 software system (GraphPad Prism Software Inc., San Diego, CA, USA), by using one-way ANOVA with Tukey's test (post hoc multiple comparisons in distributions not-departing from normality). A p-value of <0.05 was considered significant.

Conclusions
The presented research demonstrates the diverse, cell-line-dependent pro-oxidative and pro-apoptotic activity of novel pyrazolo [4,3-e]tetrazolo [1,5-b] [1,2,4]triazine sulfonamides, such as MM129, MM130, and MM131, on HeLa, PC-3, HCT 116, and BxPC-3 cells. The results from the H2DCFDA and apoptotic assays (Annexin V binding assay, Mito-Tracker assay, and AO/EB) proved the high pro-oxidative and pro-apoptotic activity of MM129, MM130, and MM131 in vitro. All investigated compounds also affected the cell cycle distribution inducing the cell-line-dependent cycle arrest in the G0/G1 or S phase. Our previous data have shown the high genotoxic potential of examined compounds toward tested cancer cells [63]. Therefore, we consider that the increase in apoptosis and the changes in the cell cycle distribution result from the strong genotoxicity of MM129, MM130, and MM131. Furthermore, based on computational studies, we conclude that the ability of the investigated sulfonamides to change the distribution of the cell cycle may result from their potential for CDKs inhibition. Moreover, we suggest that a high level of ROS may enhance the genotoxic and pro-apoptotic properties of the investigated triazine derivatives (Figure 9). Figure 9. The potential mechanism of action of MM129, MM130, and MM131. Investigated sulfonamides cause DNA single-strand and double-strand breaks and simultaneously inhibit CDKs. High levels of DNA damage and inhibition of CDKs lead to cell cycle arrest. As the effect of high genotoxic stress and the action of the G1/S cell cycle checkpoint, the cell is directed to the apoptosis pathway. The consequences of apoptosis include cell morphological changes and the exposition of PS on the outer plasma membrane. Furthermore, apoptotic changes cause loss of ΔѰm and lead to the opening of PTP, which results in ROS formation. The continuous accumulation of ROS may enhance genotoxic stress and apoptosis, leading to a self-perpetuating cycle of changes. Figure 9. The potential mechanism of action of MM129, MM130, and MM131. Investigated sulfonamides cause DNA single-strand and double-strand breaks and simultaneously inhibit CDKs. High levels of DNA damage and inhibition of CDKs lead to cell cycle arrest. As the effect of high genotoxic stress and the action of the G1/S cell cycle checkpoint, the cell is directed to the apoptosis pathway. The consequences of apoptosis include cell morphological changes and the exposition of PS on the outer plasma membrane. Furthermore, apoptotic changes cause loss of ∆Ψm and lead to the opening of PTP, which results in ROS formation. The continuous accumulation of ROS may enhance genotoxic stress and apoptosis, leading to a self-perpetuating cycle of changes.
In conclusion, the obtained data combined with the results from our previous study [63] provide important information on the biological response of the tested cancer cell lines to MM129, MM130, and MM131. Moreover, our results expand the general knowledge about the antineoplastic activity of a group of MM compounds. Interestingly, among examined sulfonamides, MM129 at the IC 50 exhibited the highest apoptotic properties in the Annexin V binding assay against HeLa cells following the 48 h incubation period ( Figure 2). Moreover, the same dose of MM129 induced the highest accumulation of cells in the subG1 phase in the case of HeLa and PC-3 cells after the 24 h of compound exposure, compared to other triazine derivatives (Figure 7). In addition, MM129 has presented the lowest binding energy values when docked against CDK enzymes, compared to MM130 and MM131 (Table 1). In contrast, MM131 at the IC 50 has revealed the highest pro-oxidative activity among examined sulfonamides towards all types of cancer cells following the 2 h of incubation time ( Figure 6). However, our results suggest that, except for those differences, the anticancer activity of tested MM compounds seems to be relatively similar. The general comparison of the anticancer activity of the tested sulfonamides based on the IC 50 and 24 h and 48 h incubation times is demonstrated in the Supplementary Material (Table S2). Undoubtedly, all the investigated triazine derivatives exhibit high antineoplastic properties. Nevertheless, despite the solid base, further investigations of the chemotherapeutic potential of MM129, MM130, and MM131 towards cervical cancer, colorectal carcinoma, prostate cancer, and pancreatic adenocarcinoma, especially on in vivo material, are required.