Ibulocydine sensitizes human cancers to radiotherapy by induction of mitochondria-mediated apoptosis
Abstract
Background and Purpose
The escalating challenge of treating human cancers, particularly in advanced stages, continuously drives the search for novel therapeutic strategies that can enhance the effectiveness of established modalities like radiotherapy. Ibulocydine (IB), a recently developed prodrug, has garnered significant attention due to its innovative mechanism of action as a cyclin-dependent kinase (CDK) inhibitor. Preliminary research has already highlighted its promising anti-cancer properties, notably demonstrated in human hepatoma cell lines. Building upon these initial observations, the overarching purpose of the current study was specifically designed to rigorously investigate the feasibility of ibulocydine as a potent radiosensitizer. The core objective was to determine whether its integration into existing treatment paradigms could significantly improve the overall efficacy of radiotherapy in combating various human cancers, thereby potentially offering a new avenue for enhancing therapeutic outcomes.
Materials and Methods
To comprehensively evaluate the radiosensitizing effects of ibulocydine, a multi-faceted experimental approach was employed, utilizing both *in vitro* cellular models and an *in vivo* animal model. Human cancer cells derived from lung and colon malignancies were meticulously treated either with ibulocydine alone, with radiotherapy (RT) alone, or with a combined regimen of both ibulocydine and radiotherapy. The cellular responses to these treatments were then extensively assessed using a suite of established biological assays. Cell viability was quantitatively determined using the CCK-8 assay, providing a measure of metabolic activity and cell count. The long-term survival and clonogenic potential of the cells were rigorously evaluated through clonogenic assays, which assess the ability of single cells to form colonies after treatment. Further insights into cellular events, such as apoptosis and cell cycle progression, were gained through flow cytometric analysis. The molecular changes at the protein level, indicative of specific cellular pathways being affected, were analyzed using Western blotting assays. Finally, to translate the *in vitro* findings into a more clinically relevant context, the *in vivo* radiotherapeutic efficacy of the combined treatment was rigorously evaluated utilizing a human cancer xenograft mouse model, which mimics tumor growth in a living system.
Results
The results unequivocally demonstrated a synergistic therapeutic benefit when ibulocydine was combined with radiotherapy. The combined treatment regimen of ibulocydine and radiotherapy led to a statistically significant reduction in both the viability and the survival fraction of the treated cancer cells, indicating a powerful anti-proliferative and cytotoxic effect. Further molecular and cellular investigations revealed that this enhanced efficacy was accompanied by a clear induction of apoptotic cell death. This programmed cell death pathway was characterized by the distinct activation of caspases, a family of executioner proteases central to apoptosis. Concurrently, a noticeable decrease in the expression ratio of Bcl-2 to Bax, pivotal regulators of the intrinsic apoptotic pathway, was observed, indicating a shift towards pro-apoptotic signaling. Moreover, a critical event in this process was the pronounced loss of mitochondrial membrane potential (MMP), which is a key hallmark of mitochondrial dysfunction and commitment to apoptosis. This loss of MMP subsequently led to the release of cytochrome c from the mitochondria into the cytosol, a crucial step that triggers the activation of downstream caspases and progression of apoptosis. To further confirm the direct involvement of the Bcl-2 protein in this pathway, an ingenious experiment was conducted: the re-expression of Bcl-2 by introducing a Bcl-2 expressing plasmid DNA into the cancer cells effectively compromised, or partially reversed, both the loss of MMP and the induction of apoptosis that had been initially observed following treatment with ibulocydine and radiotherapy. This genetic rescue experiment provided compelling evidence for the central role of Bcl-2 in mediating the observed apoptotic effects. Finally, the therapeutic efficacy of the combined treatment regimen was robustly verified in the *in vivo* xenograft mouse model. In mice treated with radiotherapy in conjunction with ibulocydine, a marked and statistically significant delay in tumor growth was observed, underscoring the radiosensitizing potential of ibulocydine in a living system.
Conclusions
In conclusion, the comprehensive findings from this study definitively demonstrate that ibulocydine possesses a remarkable property of sensitizing human cancer cells to the effects of radiotherapy. This enhanced radiosensitivity is primarily mediated through the potent induction of mitochondria-mediated apoptosis within the cancer cells. The clear molecular and cellular mechanisms, including caspase activation, modulation of Bcl-2/Bax expression, and the loss of mitochondrial membrane potential leading to cytochrome c release, collectively highlight the precise pathway through which ibulocydine enhances the therapeutic efficacy of radiation. These compelling results strongly suggest that ibulocydine is a highly promising candidate that merits further clinical investigation and consideration for its application in chemoradiotherapy regimens, offering a potential strategy to improve outcomes for patients battling various human cancers.
Keywords: Apoptosis; Bcl-2; Caspase; Ibulocydine; Mitochondria; Radiotherapy.
Introduction
Radiotherapy plays an undeniably crucial role as a primary modality in the local treatment of solid tumors. Its therapeutic efficacy is fundamentally rooted in its ability to induce significant DNA damage within cancer cells, which subsequently triggers essential cellular responses such as cell cycle arrest and, ultimately, programmed cell death or apoptosis. However, despite its widespread application and recognized importance, the full therapeutic potential of radiotherapy as a major treatment for human cancers is frequently constrained by the phenomenon of radioresistance, which manifests in many types of malignancies. This inherent resistance in cancer cells appears to arise through a diverse array of complex molecular mechanisms. These mechanisms include, but are not limited to, a persistently high and dysregulated activity of the Akt signaling pathway, the overexpression of key anti-apoptotic proteins such as Bcl-2 and Bcl-xL, and various defects in the proper release of mitochondrial proteins that are crucial for initiating apoptosis. Furthermore, the elevated expression of inhibitors of apoptosis proteins (IAPs), such as survivin and XIAP, can also significantly contribute to radioresistance, as these proteins directly block the apoptotic cascade at its effector phase, thereby preventing cell death. Given these pervasive challenges, an intensive and ongoing scientific endeavor is currently focused on identifying novel radiosensitizers—compounds capable of effectively overcoming this intrinsic radioresistance in cancer cells, thereby enhancing the therapeutic window of radiotherapy. Among the promising candidates in this pursuit are several cyclin-dependent kinase (CDK) inhibitors, including flavopiridol, roscovitine, SNS-032, and AZD5438. These agents have demonstrated both intrinsic anti-cancer activity and a remarkable ability to increase the cellular sensitivity of various human cancer cells to both radiation and a range of chemotherapeutic drugs.
The cyclin-dependent kinases (CDKs) are universally recognized as fundamental and indispensable regulators of cell cycle progression, orchestrating the precise timing and sequence of cellular division through their dynamic association with specific cyclins. The aberrant deregulation of CDK activation or the pervasive overexpression of cyclins are frequently observed molecular hallmarks in a vast array of human cancers, contributing directly to uncontrolled cellular proliferation. Given that uncontrolled cell growth is a defining characteristic and a hallmark of neoplastic cells, the targeted inhibition of CDKs has emerged as a potent and highly attractive strategy for cancer treatment. Ibulocydine (IB), the focus of this investigation, is an isobutyrate ester prodrug of a novel synthetic CDK inhibitor. It exhibits specific activity against CDK7 and CDK9, two kinases with critical roles in transcriptional regulation. As detailed in our previous report, ibulocydine has already demonstrated significant antiproliferative and antitumoral effects in human hepatocellular carcinoma cells. Mechanistically, ibulocydine was found to induce apoptosis through a rapid downregulation of anti-apoptotic proteins, further underscoring its potential as an anti-cancer agent.
In the present study, our primary objective was to thoroughly investigate the therapeutic effects of ibulocydine when administered in combination with radiotherapy in various human cancer cell lines. This comprehensive assessment aimed to critically evaluate the potential for ibulocydine’s clinical application as a radiosensitizer. We have conclusively demonstrated that radiotherapy, when combined with even low doses of ibulocydine, significantly induced apoptotic cell death. This process was found to occur through the distinct activation of caspases, a cascade initiated by the loss of mitochondrial membrane potential (MMP) and the subsequent release of cytochrome c into the cytosol, driven by the crucial downregulation of the Bcl-2/Bax protein ratio. These findings strongly suggest that ibulocydine possesses a robust capacity to act as a radiosensitizer, making it a promising candidate for clinical application in the context of chemoradiotherapy for human cancers.
Materials and methods
Reagents
For the comprehensive cellular and molecular analyses conducted in this study, a diverse array of high-quality antibodies and other essential reagents were meticulously sourced. Antibodies targeting PARP and cytochrome c were obtained from BD Biosciences Pharmingen, while those against caspase-9 were from Upstate, and caspase-8 from Millipore. Bcl-2 antibodies were from Invitrogen, Mcl-1 from Calbiochem, and Flag and beta-actin from Sigma. VDAC and survivin antibodies were purchased from Santa Cruz Biotechnology. A more extensive panel of antibodies, including those for caspase-3, cleaved caspase-3, Bax, Bcl-xL, cIAP1, Bad, Bid, XIAP, and cIAP2, were obtained from Cell Signaling Technology. Antibodies against p-RNA pol II (Ser-2), p-RNA pol II (Ser-5), NOXA, and RNA pol II were procured from Abcam. Additionally, secondary antibodies, including rabbit IgG horseradish peroxidase, mouse IgG, and goat IgG, were sourced from Zymed Laboratories, Inc., ensuring appropriate detection in Western blotting.
Culture of Cancer Cells
To conduct the *in vitro* experiments, several established human cancer cell lines were utilized, each maintained under specific optimal culture conditions to ensure their viability and proliferation. Human lung cancer A549 cells were cultured in F-12 K medium, which was supplemented with 10% fetal bovine serum (FBS) and a standard cocktail of antibiotics (Life Technologies) to prevent microbial contamination. Human colon cancer RKO cells and human hepatocellular carcinoma Hep3B cells were maintained in MEM (Minimum Essential Medium) containing 10% FBS and antibiotics (Life Technologies). Lastly, human breast cancer MCF-7 cells were grown in RPMI (Roswell Park Memorial Institute) 1640 medium, also supplemented with 10% FBS and antibiotics (Life Technologies). All cell lines were cultured in a humidified atmosphere with 5% CO2 at 37 degrees Celsius.
Cell Viability Assay and Clonogenic Assay
To quantitatively assess the cytotoxic impact of ibulocydine (IB) and radiotherapy (RT), a series of robust cellular assays were performed. Cytotoxicity was initially determined using the Cell Counting Kit-8 (CCK-8) assay, meticulously following the manufacturer’s protocol. This colorimetric assay measures cellular metabolic activity, providing a reliable proxy for cell viability. For the clonogenic assay, which assesses the long-term reproductive viability of cells, cells were carefully seeded into a 6-well plate. Following a 2-hour pretreatment with IB at specified concentrations (0.01 or 0.1 μM), the cells were then exposed to different doses of RT. After irradiation, the cells were incubated for a period of 9 days, allowing sufficient time for surviving cells to form colonies. Subsequently, the colonies were stained with a 0.5% crystal violet solution in 10% methanol. Only colonies comprising more than 50 cells were counted, and the survival fraction for each treatment group was normalized to the surviving fraction obtained from the untreated control cells, providing a standardized measure of cell survival after treatment.
Establishment of the Stable Cell Lines Overexpressing Bcl-2
To precisely investigate the role of Bcl-2 in the observed cellular responses, stable cell lines capable of overexpressing Bcl-2 were established. Human lung cancer A549 and human colon cancer RKO cells were genetically modified by transfection with either a Flag-tagged backbone vector (plasmid 3261) or a plasmid containing the full-length bcl-2 cDNA, which was kindly provided by Dr. A. Strasser from The Walter and Eliza Hall Institute of Medical Research. Following transfection, the cells were subjected to selection in fresh culture media containing 4 micrograms per milliliter of puromycin, ensuring that only cells stably expressing the introduced plasmid survived and proliferated. The successful overexpression of Bcl-2 in these newly established stable cell lines was rigorously verified through Western blotting, utilizing both a Flag-antibody to detect the tagged protein and a specific Bcl-2-antibody to confirm its presence and expression level.
Annexin V-Fluorescein and PI Staining
To detect and quantify the induction of apoptosis, cells were subjected to Annexin V-Fluorescein and propidium iodide (PI) double staining, a widely accepted method for identifying early and late apoptotic cells. Cells were first gently digested with trypsin to detach them from the culture surface and then washed twice with phosphate-buffered saline (PBS) to remove any residual trypsin. The staining procedure was performed using the Annexin-V-FLUOS Staining Kit (Roche), strictly adhering to the manufacturer’s manual. Briefly, cells were stained with 5 microliters of Annexin-V-FLUOS, which binds to externalized phosphatidylserine on apoptotic cell membranes, and 1 microliter of propidium iodide (PI) staining solution, a DNA intercalating dye that enters cells with compromised membranes (late apoptotic/necrotic cells). The staining incubation was carried out in the dark at room temperature for 15 minutes. Following staining, the cell samples were immediately analyzed by flow cytometry (Becton-Dickinson), allowing for the quantitative assessment of apoptotic and necrotic cell populations based on their specific fluorescent signals.
Cell Cycle Analysis
To ascertain the impact of treatments on cell cycle progression, cell cycle analysis was performed. Cells, including both those adherent to the culture surface (after trypsinization) and any floating cells, were meticulously pooled together. They were then washed thoroughly with PBS–EDTA solution to ensure a clean suspension and subsequently fixed in 70% (v/v) ice-cold ethanol, a standard fixative that preserves DNA content. After fixation, the DNA content of the cells was precisely assessed by staining them with propidium iodide (PI), a fluorescent dye that quantitatively binds to DNA. The PI-stained cells were then analyzed by flow cytometry, which measures the fluorescence intensity of individual cells, directly reflecting their DNA content. The distribution of cells within different phases of the cell cycle (G0/G1, S, and G2/M) was accurately determined using the specialized ModFit LT program (Verity Software House).
In Vivo Tumor Growth Delay
To assess the *in vivo* radiosensitizing effect of ibulocydine, a xenograft tumor model was established using human lung cancer A549 cells. A precisely prepared suspension of 1 x 10^6 A549 cells, contained in a 50 microliter volume, was subcutaneously injected into the right hind limb of male Balb/c nude mice, which were 5 weeks old and obtained from SLC. Tumors were allowed to grow until their average volume reached approximately 70-80 cubic millimeters. Once the target tumor size was achieved, the mice were systematically divided into distinct treatment groups, with each group comprising 4 mice, ensuring adequate statistical power. The treatment regimen commenced with the intratumoral injection of ibulocydine (IB) at a dose of 100 micrograms per mouse. Two hours following the IB injection, tumors were precisely irradiated with a single dose of 2 Gy using a 6-MV photon beam linear accelerator (CL/1800, Varian Medical System Inc.). The combined treatment of IB and radiotherapy was repeated three times, with a three-day interval between each administration. The volume of the tumors was diligently measured at regular intervals and calculated using the established formula: V = (L x W^2) x 0.5, where V represents volume, L denotes the longest dimension (length), and W denotes the perpendicular width.
Statistical Analysis and Determination of Synergy
To ensure the robust and reliable interpretation of all experimental data, statistical analysis was performed. All data obtained were consistently presented as the mean value plus or minus the standard error (mean ± SE) from at least three independent experiments, ensuring reproducibility. The statistical analyses were conducted using SPSS v12.0.1 software (SPSS). Mean values were rigorously compared using unpaired T-tests for two-group comparisons and analysis of variance (ANOVA) for comparisons involving multiple groups, followed by Bonferroni correction for multiple comparisons to maintain statistical rigor. For all analyses, a P-value of less than 0.05 (P < 0.05) was uniformly considered to represent a statistically significant difference. The potential synergistic effect of ibulocydine (IB) and radiotherapy (RT) was critically evaluated using the well-established isobologram method. Briefly, cells were treated with different concentrations of IB and RT, either alone or in combination. After a 24-hour incubation period, the relative survival of the cells was assessed. The generated concentration-effect curves were then utilized to determine the IC50 (half-maximal inhibitory concentration) values for each agent administered alone and for the combination treatment with a fixed concentration of the second agent, thereby allowing for the graphical and quantitative assessment of synergy.
Supplementary Information
Detailed methodologies for additional experiments, including the precise measurement of mitochondrial membrane potential (MMP), the meticulous isolation of cytosolic fractions, the comprehensive analysis of mitochondrial protein release, quantitative real-time PCR (qPCR) analysis, and Western blot analysis, are comprehensively provided in the Supplementary Information section. This ensures transparency and reproducibility of all experimental procedures.
Results
To quantitatively assess the cytotoxic efficacy of ibulocydine (IB) on human lung cancer A549 cells and human colon cancer RKO cells, these cells were systematically treated with varying doses of IB for a 24-hour period. The viability of the cells was notably reduced by IB, with significant effects observed at doses of 1 μM for A549 cells and above 0.8 μM for RKO cells. While radiotherapy (RT) alone, even at doses up to 10 Gy, induced only a limited amount of cell death (less than 10%) over 24 hours, the synergistic effect was striking. RT, when administered in the presence of IB, significantly decreased cell viability in both A549 and RKO cell lines. Furthermore, similar synergistic cell death was observed when combining IB and RT in other human cancer cell lines, specifically human hepatocellular carcinoma Hep3B cells and human breast cancer MCF-7 cells. Moreover, a rigorous isobologram analysis definitively demonstrated synergistic interactions between IB and RT across all tested cell lines: A549, RKO, Hep3B, and MCF-7. The impact of IB on the long-term survival of the cells subjected to RT was precisely determined by clonogenic assay. This assay revealed that the survival fraction of cells was markedly decreased by the combination of IB with RT in both A549 and RKO cells. These collective results powerfully demonstrated that a sub-lethal dose of ibulocydine efficiently sensitized human cancer cells to the cytotoxic effects of radiotherapy, highlighting its potential as a radiosensitizer.
We then thoroughly characterized the specific type of cell death associated with radiosensitization by ibulocydine. The changes in DNA content, indicative of apoptosis, were meticulously examined following treatment with IB and/or RT. Co-treatment of IB and RT for 24 hours led to a significant increase in the accumulation of cells in the sub-G1 phase, a hallmark of apoptotic DNA fragmentation, whereas treatment with IB or RT alone did not produce such a pronounced effect. Further confirmation came from the annexin V/PI double-staining assay, which detects phosphatidylserine externalization, an early event in apoptosis. While cells treated with IB or RT alone showed only a small proportion of annexin V-positive cells, a significantly larger number of apoptotic cells were observed when IB and RT were combined. These results unequivocally indicated that the combined treatment of IB and RT dramatically increased the induction of apoptosis compared to either single treatment, confirming the synergistic induction of programmed cell death.
Next, we investigated whether the augmented apoptosis induced by IB was dependent on caspase activation, a crucial cascade in programmed cell death. The results clearly revealed that neither IB nor RT alone induced the proteolytic processing of any caspases or caspase-related proteins in either cell line. In striking contrast, the combined treatment with IB and RT specifically induced the cleavage and activation of caspase-3 and caspase-9, as well as the cleavage of PARP, a known substrate protein of caspase-3, confirming the activation of the intrinsic apoptotic pathway. Interestingly, the precursor protein levels of caspase-8, typically involved in the extrinsic apoptotic pathway, did not decrease in any of the treatment conditions, suggesting that the intrinsic pathway is primarily engaged. To further solidify the role of caspases, pre-treatment of cells with the pan-caspase inhibitor zVAD significantly blocked the cell death induced by the combined treatment. Furthermore, pre-treatment with zVAD also markedly inhibited the reduction of colony formation induced by the combined treatment in both A549 and RKO cells, providing functional evidence. These collective results strongly suggested that caspases played an indispensable and critical role in the apoptosis induced by the combined treatment of ibulocydine and radiotherapy.
To further confirm the direct effect of ibulocydine on its purported targets, Cdk7 and Cdk9, we precisely measured the cellular levels of RNA polymerase II phosphoforms at Ser-2 or Ser-5. These phosphorylation sites on RNA polymerase II are specifically regulated by Cdk9 and Cdk7, respectively, serving as direct indicators of their activity. The results unequivocally indicated that ibulocydine effectively reduced the phosphorylation of RNA polymerase II at both Ser-2 and Ser-5 in a clear dose-dependent manner, confirming its inhibitory action on Cdk7 and Cdk9.
To comprehensively understand the intricate underlying mechanisms by which ibulocydine effectively sensitizes human cancer cells to radiotherapy through the induction of apoptosis, we systematically investigated the modulations of various key apoptosis regulators in response to ibulocydine treatment. As explicitly shown, the expression of the anti-apoptotic Bcl-2 protein was markedly decreased, while the pro-apoptotic Bax protein was concomitantly increased by ibulocydine in a dose-dependent manner, indicating a crucial shift in the balance of these regulatory proteins. The levels of other apoptosis-related proteins investigated, however, remained unaltered in both cell lines. Consistently, with the observed changes at the protein level, ibulocydine also caused a decrease in Bcl-2 mRNA and a corresponding increase in Bax mRNA, suggesting that ibulocydine modulates the expression of Bcl-2 or Bax primarily at the transcriptional level. Furthermore, treatment with both ibulocydine and radiotherapy dramatically altered the Bcl-2/Bax expression ratio in both cell lines, whereas radiotherapy alone did not induce such a pronounced effect. These consistent results strongly implied that the specific regulation of Bcl-2/Bax by ibulocydine was centrally involved in its potent radiosensitizing effect, driving the cells towards an apoptotic fate.
Bcl-2 and Bax are pivotal regulators that precisely control mitochondrial membrane potential (MMP), and the loss of MMP plays an indispensable role in the initiation of apoptosis, leading to the crucial release of key apoptosis mediators such as cytochrome c, which subsequently triggers the activation of downstream apoptosis signaling cascades. Therefore, we meticulously examined whether an increased activation of the mitochondrial pathway actively contributed to the enhancement of radiotherapy-induced apoptosis by ibulocydine. Changes in MMP were quantitatively measured by flow cytometry analysis, and the release of cytochrome c into the cytosol was precisely analyzed by Western blotting. The results revealed that while treatment with ibulocydine or radiotherapy alone mediated only a slight loss of MMP, the combined treatment of ibulocydine and radiotherapy provoked a significantly more pronounced and dramatic loss of MMP, indicating a synergistic effect on mitochondrial integrity. Accordingly, the release of cytochrome c into the cytosol was immensely increased by the combined treatment of ibulocydine and radiotherapy, providing direct evidence of mitochondrial outer membrane permeabilization. Importantly, in cells where Bcl-2 expression was restored and recovered by introducing a Bcl-2 expressing plasmid, the characteristic loss of MMP, the reduction of colony formation, and the induction of apoptosis previously observed with ibulocydine and radiotherapy treatment were significantly compromised. This compelling genetic rescue experiment strongly suggested that the decrease in Bcl-2 expression was critically involved in the signal transduction leading to radiosensitization. Taken together, these comprehensive findings unequivocally demonstrated that ibulocydine sensitized human cancer cells to radiotherapy primarily by effectively triggering the mitochondrial apoptotic pathway.
To comprehensively assess whether ibulocydine exerts a significant *in vivo* radiosensitizing effect, xenograft mice bearing tumors derived from human A549 cells were meticulously subjected to a controlled *in vivo* experiment. The results indicated that treatment with either radiotherapy alone or ibulocydine alone did not induce a substantial or statistically significant delay in tumor growth. However, the combined treatment of ibulocydine and radiotherapy consistently displayed a remarkable and statistically significant therapeutic effect, leading to a profound delay in tumor growth. Crucially, this enhanced anti-tumor efficacy was achieved without any notable loss of body weight in the treated mice, indicating a favorable therapeutic window and tolerability. It was particularly noteworthy that the tumor growth in mice treated with radiotherapy in conjunction with ibulocydine was almost completely halted for a period of 15 days, a duration that included the initial 9-day administration period. Subsequent Western blot analyses performed on tumor specimens obtained from the mice revealed a significant decrease in the protein levels of Bcl-2 within the combined-treatment group when compared to the control groups. This molecular change in Bcl-2 expression *in vivo* further supported the hypothesis that ibulocydine exerts its radiosensitization effect through a Bcl-2-dependent mitochondrial and caspase pathway, thereby translating its *in vitro* mechanisms into a tangible *in vivo* therapeutic benefit.
Discussion
Radiotherapy, despite its foundational role as one of the most effective modalities in the clinical treatment of cancer, frequently falls short of achieving a completely satisfactory curative effect in all patients. The ultimate therapeutic influence of radiotherapy in cancer patients is largely contingent upon two critical factors: the inherent radiosensitivity of the tumor itself and the tolerance limits of surrounding normal tissues, which dictate the maximum tolerable radiation dose. Furthermore, solid tumors often present an additional challenge, as their inherent hypoxic regions tend to significantly lessen the efficacy of radiotherapy, contributing to treatment resistance. To address these limitations and substantially improve the overall therapeutic outcomes of radiotherapy, there is an urgent and critical need for the discovery of novel drugs that can act as potent radiosensitizers, effectively disrupting tumor growth pathways in concert with radiation-induced damage. The clinical application of such innovative compounds is therefore of paramount importance. Building upon the compelling evidence generated in this study, we are now inceptively proposing that ibulocydine (IB) is indeed a highly promising candidate that is well worthwhile to be developed as an effective radiosensitizer and subsequently applied for human cancer treatment. Our comprehensive investigation has demonstrably shown that even low doses of ibulocydine, when combined with radiotherapy, can significantly improve radiotherapeutic efficacy through the potent induction of caspase-dependent apoptosis. Furthermore, we have precisely elucidated the underlying mechanisms of IB-mediated radiosensitization, revealing that the crucial loss of mitochondrial membrane potential (MMP) and the subsequent release of cytochrome c, resulting from the intricate regulations of Bcl-2/Bax proteins, collectively contribute to the activation of the downstream caspase cascades, thereby driving the cells towards programmed death.
We previously reported that ibulocydine, a novel inhibitor specifically targeting Cdk7 and Cdk9, exhibited a distinct mechanism of action wherein it caused a rapid downregulation of various anti-apoptotic proteins, consequently inducing apoptosis in human hepatocellular carcinoma cells. This finding is paralleled by other studies, such as that by Chen and colleagues, who reported SNS-032, another inhibitor of Cdk2/7/9, to effectively decrease the mRNA and protein levels of anti-apoptotic proteins in chronic lymphocytic leukemia. Cdk7 and Cdk9 have been extensively studied and are widely recognized as key regulators of RNA polymerase II regulation, playing indispensable roles in both the initiation and elongation steps of gene transcription. For instance, Cdk7 is an integral and essential component of the transcription factor TFIIH, a multiprotein complex crucial for transcription. Cdk7 specifically phosphorylates the Ser-5 residues within the heptad repeats of the C-terminal domain (CTD) of RNA polymerase II (Pol II), a phosphorylation event that is critical to facilitate transcription initiation. Conversely, Cdk9, which forms a vital part of the elongation factor P-TEFb, performs a complementary function by phosphorylating Ser-2 residues in the CTD of RNA Pol II, a modification that is strictly required for efficient transcript elongation. Despite the well-established roles of CDK7 and CDK9 in RNA polymerase regulation, the precise and accurate mechanisms by which their inhibition acts as a target for anti-cancer therapy have, until now, remained somewhat unclear. Nonetheless, our present study has definitively demonstrated the feasibility of ibulocydine as a clinically applicable radiosensitizer by thoroughly elucidating the underlying molecular mechanisms through which it exerts its profound radiosensitization effect.
Apoptosis, a fundamental and highly regulated mechanism of programmed cell death, can be driven primarily by two major apoptotic pathways: the cell death receptor-mediated extrinsic pathway and the mitochondrial-mediated intrinsic pathway. The mitochondrial-mediated apoptotic pathway is characterized by a series of critical events, including the discernible loss of mitochondrial membrane potential (MMP), the subsequent release of cytochrome c from the mitochondria into the cytosol, and the ultimate proteolytic cleavage and activation of executioner caspase-3. These sequential events culminate in the characteristic morphological and biochemical changes of apoptosis, such as chromatin condensation, DNA fragmentation, and the formation of apoptotic bodies. The Bcl-2 family members, specifically anti-apoptotic Bcl-2 and pro-apoptotic Bax, are pivotal proteins that intricately regulate apoptosis. An altered balance, particularly overexpression of pro-apoptotic Bax and low expression of anti-apoptotic Bcl-2, often leads to the collapse of MMP, the release of cytochrome c, and the activation of caspase-3, either through homologous dimerization or by promoting the formation of mitochondrial permeability transition pores in the inner and outer membranes. These proteins are predominantly localized in the mitochondrion and are considered to be directly associated with the crucial release of cytochrome c. Our comprehensive results clearly showed that treatment with ibulocydine and radiotherapy synergistically reduced the cellular level of anti-apoptotic Bcl-2 while concomitantly increasing the levels of pro-apoptotic Bax. Consequently, this molecular shift profoundly induced the loss of MMP and the release of cytochrome c into the cytosol, robustly indicating that significant mitochondrial dysfunction was critically involved in ibulocydine-induced apoptosis when combined with radiotherapy. To further and definitively confirm a causal impact of Bcl-2 in ibulocydine-mediated radiosensitization, we meticulously established Bcl-2-overexpressing A549 and RKO cells. Our subsequent experiments unequivocally determined that the overexpression of Bcl-2 significantly blocked the ibulocydine-induced apoptosis and the loss of MMP upon radiotherapy, providing compelling genetic evidence for Bcl-2’s central role in mediating the radiosensitization process.
An intriguing and particularly significant finding emerged regarding the signal transduction underlying ibulocydine-mediated radiosensitization. While treatment with ibulocydine alone did induce an alteration in the protein levels of Bcl-2 and Bax, likely with an effect that was only slightly weaker than that observed with the combined treatment of ibulocydine and radiotherapy, it was striking that ibulocydine alone did not appropriately induce the pronounced loss of MMP, the significant release of cytochrome c, or the consequent widespread cell death. A plausible explanation for this crucial distinction is that the irreversible loss of MMP, leading to profound cell death, is triggered only in the specific circumstance where both radiotherapy-mediated signal transduction pathways are active and simultaneously coupled with ibulocydine-mediated reduction of Bcl-2. This suggests a critical interplay: the reduction of Bcl-2 by ibulocydine is a necessary condition, but it is not by itself sufficient to fully induce radiosensitization; a synergistic signal from radiation is also required to fully unleash the apoptotic cascade.
In conclusion, we have herein robustly demonstrated that ibulocydine effectively sensitizes human cancer cells to radiotherapy. This radiosensitization is mechanistically driven by a cascade of events including the crucial release of cytochrome c, a significant loss of mitochondrial membrane potential, and a beneficial downregulation of the anti-apoptotic Bcl-2/Bax expression ratio. Moreover, Atuveciclib the therapeutic potential of ibulocydine was further solidified by its ability to significantly delay tumor growth when combined with radiotherapy in the A549-derived xenograft tumor model *in vivo*. These compelling findings collectively suggest that ibulocydine stands as a highly promising candidate that warrants further clinical investigation and consideration for its potential clinical application as an effective radiosensitizer in comprehensive cancer treatment regimens.