Duocarmycin SA Reduces Proliferation and Increases Apoptosis in Acute Myeloid Leukemia Cells In Vitro.

Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by an expansion of immature myeloid precursors. Despite therapeutic advances, the prognosis of AML patients remains poor and there is a need for the evaluation of promising therapeutic candidates to treat the disease. The objective of this study was to evaluate the efficacy of duocarmycin Stable A (DSA) in AML cells in vitro. We hypothesized that DSA would induce DNA damage in the form of DNA double-strand breaks (DSBs) and exert cytotoxic effects on AML cells within the picomolar range. Human AML cell lines Molm-14 and HL-60 were used to perform 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), DNA DSBs, cell cycle, 5-ethynyl-2-deoxyuridine (EdU), colony formation unit (CFU), Annexin V, RNA sequencing and other assays described in this study. Our results showed that DSA induced DNA DSBs, induced cell cycle arrest at the G2M phase, reduced proliferation and increased apoptosis in AML cells. Additionally, RNA sequencing results showed that DSA regulates genes that are associated with cellular processes such as DNA repair, G2M checkpoint and apoptosis. These results suggest that DSA is efficacious in AML cells and is therefore a promising potential therapeutic candidate that can be further evaluated for the treatment of AML.

GA-OH enhances the cytotoxicity of photon and proton radiation in HPV+ HNSCC cells.

Introduction: Treatment-related toxicity following either chemo- or radiotherapy can create significant clinical challenges for HNSCC cancer patients, particularly those with HPV-associated oropharyngeal squamous cell carcinoma. Identifying and characterizing targeted therapy agents that enhance the efficacy of radiation is a reasonable approach for developing de-escalated radiation regimens that result in less radiation-induced sequelae. We evaluated the ability of our recently discovered, novel HPV E6 inhibitor (GA-OH) to radio-sensitize HPV+ and HPV- HNSCC cell lines to photon and proton radiation. Methods: Radiosensitivity to either photon or proton beams was assessed using various assays such as colony formation assay, DNA damage markers, cell cycle and apoptosis, western blotting, and primary cells. Calculations for radiosensitivity indices and relative biological effectiveness (RBE) were based on the linear quadratic model. Results: Our results showed that radiation derived from both X-ray photons and protons is effective in inhibiting colony formation in HNSCC cells, and that GA-OH potentiated radiosensitivity of the cells. This effect was stronger in HPV+ cells as compared to their HPV- counterparts. We also found that GA-OH was more effective than cetuximab but less effective than cisplatin (CDDP) in enhancing radiosensitivity of HSNCC cells. Further tests indicated that the effects of GA-OH on the response to radiation may be mediated through cell cycle arrest, particularly in HPV+ cell lines. Importantly, the results also showed that GA-OH increases the apoptotic induction of radiation as measured by several apoptotic markers, even though radiation alone had little effect on apoptosis. Conclusion: The enhanced combinatorial cytotoxicity found in this study indicates the strong potential of E6 inhibition as a strategy to sensitize cells to radiation. Future research is warranted to further characterize the interaction of GA-OH derivatives and other E6-specific inhibitors with radiation, as well as its potential to improve the safety and effectiveness of radiation treatment for patients with oropharyngeal cancer.

Proton-induced DNA damage promotes integration of foreign plasmid DNA into human genome.

High-risk human papillomaviruses (HPVs) cause virtually all cervical cancer cases and are also associated with other types of anogenital and oropharyngeal cancers. Normally, HPV exists as a circular episomal DNA in the infected cell. However, in some instances, it integrates into the human genome in such a way as to enable increased expression of viral oncogenes, thereby leading to carcinogenesis. Since viral integration requires breaks in both viral and human genomes, DNA damage likely plays a key role in this critical process. One potentially significant source of DNA damage is exposure to elevated doses of ionizing radiation. Natural background radiation is ubiquitous; however, some populations, including radiological workers, radiotherapy patients, and astronauts, are exposed to significantly higher radiation doses, as well as to different types of radiation such as particle radiation. We hypothesize that ionizing radiation-induced DNA damage facilitates the integration of HPV into the human genome, increasing the risk of developing HPV-related cancers in the exposed population. To test this, we first determined the kinetics of DNA damage in keratinocytes exposed to ionizing radiation (protons) by assessing γ-H2AX foci formation using immunofluorescence (direct damage), and also measured ROS and 8-oxoG levels via DCFDA and Avidin-FITC (indirect damage).As anticipated, direct DNA damage was observed promptly, within 30 min, whereas indirect DNA damage was delayed due to the time required for ROS to accumulate and cause oxidative damage. Although radiation was lethal at high doses, we were able to establish an experimental system where radiation exposure (protons and X-rays) induced DNA damage dose-dependently without causing major cytotoxic effects as assessed by several cytotoxicity assays. Most importantly, we explored the impact of radiation exposure on integration frequency using a clonogenic assay and demonstrated that as predicted, proton-induced DNA damage promotes the integration of HPV-like foreign DNA in oral keratinocytes. Overall, the insights gained from this work enable us to better understand the contribution of radiation exposure and DNA damage to HPV-mediated carcinogenesis and direct us toward strategies aimed at preventing malignancies in HPV-infected individuals.

Convalescent Plasma for COVID-19: A Single Center Prospective Experience with Serial Antibody Measurements and Review of the Literature.

Background: High-titer convalescent plasma given early for COVID-19 may decrease progression into a severe infection. Here, we reported a study of serial antibody measurements in patients who received CP at our center and performed a systematic review of randomized trials on CP. Methods: Our center participated in the Mayo Clinic Expanded Access Program for COVID-19 Convalescent Plasma. Patients diagnosed with COVID-19 by nasopharyngeal polymerase chain reaction at our center between April and August 2020 were included in the study if staffing was available for specimen collection. Through a colloidal gold immunochromatography assay, these patients' IgM and IgG antibody responses were measured at baseline (Day 0) and after transfusion (Day 1, 2, etc.). Donor CP antibody levels were measured as well. Results: 110 serum specimens were obtained from 21 COVID-19 patients, 16 of whom received CP. The median time from developing symptoms to receiving CP was 11 days (range 4−21). In 9 of 14 (64%) cases where both recipient and donor CP antibody levels were tested, donor COVID-19 IgG was lower than that of the recipient. Higher donor antibody levels compared with the recipient (R = 0.71, p < 0.01) and low patient IgG before CP transfusion (p = 0.0108) correlated with increasing patient IgG levels from baseline to Day 1. Among all patients, an increased COVID-19 IgG in the short-term and longitudinally was positively correlated with improved clinical outcomes (ρ = 0.69, p = 0.003 and ρ = 0.58, p < 0.006, respectively). Conclusions: In a real-world setting where donor CP was not screened for the presence of antibodies, CP in donors might have less COVID-19 IgG than in recipients. An increase in patient antibody levels in the short term and longitudinally was associated with improved clinical outcomes.

ACE2 : S1 RBD Interaction-Targeted Peptides and Small Molecules as Potential COVID-19 Therapeutics.

The COVID-19 pandemic that began in late 2019 continues with new challenges arising due to antigenic drift as well as individuals who cannot or choose not to take the vaccine. There is therefore an urgent need for additional therapies that complement vaccines and approved therapies such as antibodies in the fight to end or slow down the pandemic. SARS-CoV-2 initiates invasion of the human target cell through direct contact between the receptor-binding domain of its Spike protein and its cellular receptor, angiotensin-converting enzyme-2 (ACE2). The ACE2 and S1 RBD interaction, therefore, represents an attractive therapeutic intervention to prevent viral entry and spread. In this study, we developed a proximity-based AlphaScreen™ assay that can be utilized to quickly and efficiently screen for inhibitors that perturb the ACE2 : S1 RBD interaction. We then designed several peptides candidates from motifs in ACE2 and S1 RBD that play critical roles in the interaction, with and without modifications to the native sequences. We also assessed the possibility of reprofiling of candidate small molecules that previously have been shown to interfere with the viral entry of SARS-CoV. Using our optimized AlphaScreen™ assay, we evaluated the activity and specificity of these peptides and small molecules in inhibiting the binding of ACE2 : S1 RBD. This screen identified cepharanthine as a promising candidate for development as a SARS-CoV-2 entry inhibitor.

Targeted Therapy as a Potential De-Escalation Strategy in Locally Advanced HPV-Associated Oropharyngeal Cancer: A Literature Review.

The treatment landscape of locally advanced HPV-oropharyngeal squamous cell carcinoma (OPSCC) is undergoing transformation. This is because the high cures rates observed in OPSCC are paired with severe treatment-related, long-term toxicities. These significant adverse effects have led some to conclude that the current standard of care is over-treating patients, and that de-intensifying the regimens may achieve comparable survival outcomes with lower toxicities. Consequently, several de-escalation approaches involving locally advanced OPSCC are underway. These include the reduction of dosage and volume of intensive cytotoxic regimens, as well as elimination of invasive surgical procedures. Such de-intensifying treatments have the potential to achieve efficacy and concurrently alleviate morbidity. Targeted therapies, given their overall safer toxicity profiles, also make excellent candidates for de-escalation, either alone or alongside standard treatments. However, their role in these endeavors is currently limited, because few targeted therapies are currently in clinical use for head and neck cancers. Unfortunately, cetuximab, the only FDA-approved targeted therapy, has shown inferior outcomes when paired with radiation as compared to cisplatin, the standard radio-sensitizer, in recent de-escalation trials. These findings indicate the need for a better understanding of OPSCC biology in the design of rational therapeutic strategies and the development of novel, OPSCC-targeted therapies that are safe and can improve the therapeutic index of standard therapies. In this review, we summarize ongoing research on mechanism-based inhibitors in OPSCC, beginning with the salient molecular features that modulate tumorigenic processes and response, then exploring pharmacological inhibition and pre-clinical validation studies of candidate targeted agents, and finally, summarizing the progression of those candidates in the clinic.

DNA damage in cancer development: special implications in viral oncogenesis.

DNA lesions arise from a combination of physiological/metabolic sources and exogenous environmental influences. When left unrepaired, these alterations accumulate in the cells and can give rise to mutations that change the function of important proteins (i.e. tumor suppressors, oncoproteins), or cause chromosomal rearrangements (i.e. gene fusions) that also result in the deregulation of key cellular molecules. Progressive acquisition of such genetic changes promotes uncontrolled cell proliferation and evasion of cell death, and hence plays a key role in carcinogenesis. Another less-studied consequence of DNA damage accumulating in the host genome is the integration of oncogenic DNA viruses such as Human papillomavirus, Merkel cell polyomavirus, and Hepatitis B virus. This critical step of viral-induced carcinogenesis is thought to be particularly facilitated by DNA breaks in both viral and host genomes. Therefore, the impact of DNA damage on carcinogenesis is magnified in the case of such oncoviruses via the additional effect of increasing integration frequency. In this review, we briefly present the various endogenous and exogenous factors that cause different types of DNA damage. Next, we discuss the contribution of these lesions in cancer development. Finally, we examine the amplified effect of DNA damage in viral-induced oncogenesis and summarize the limited data existing in the literature related to DNA damage-induced viral integration. To conclude, additional research is needed to assess the DNA damage pathways involved in the transition from viral infection to cancer. Discovering that a certain DNA damaging agent increases the likelihood of viral integration will enable the development of prophylactic and therapeutic strategies designed specifically to prevent such integration, with an ultimate goal of reducing or eliminating these viral-induced malignancies.

PPI Modulators of E6 as Potential Targeted Therapeutics for Cervical Cancer: Progress and Challenges in Targeting E6.

Advanced cervical cancer is primarily managed using cytotoxic therapies, despite evidence of limited efficacy and known toxicity. There is a current lack of alternative therapeutics to treat the disease more effectively. As such, there have been more research endeavors to develop targeted therapies directed at oncogenic host cellular targets over the past 4 decades, but thus far, only marginal gains in survival have been realized. The E6 oncoprotein, a protein of human papillomavirus origin that functionally inactivates various cellular antitumor proteins through protein-protein interactions (PPIs), represents an alternative target and intriguing opportunity to identify novel and potentially effective therapies to treat cervical cancer. Published research has reported a number of peptide and small-molecule modulators targeting the PPIs of E6 in various cell-based models. However, the reported compounds have rarely been well characterized in animal or human subjects. This indicates that while notable progress has been made in targeting E6, more extensive research is needed to accelerate the optimization of leads. In this review, we summarize the current knowledge and understanding of specific E6 PPI inhibition, the progress and challenges being faced, and potential approaches that can be utilized to identify novel and potent PPI inhibitors for cervical cancer treatment.

A high-content AlphaScreen™ identifies E6-specific small molecule inhibitors as potential therapeutics for HPV+ head and neck squamous cell carcinomas.

The incidence of human papillomavirus-positive head and neck squamous cell carcinoma (HPV+-HNSCC) has increased dramatically over the past decades due to an increase in infection of the oral mucosa by HPV. The etiology of HPV+-HNSCC is linked to expression of the HPV oncoprotein, E6, which influences tumor formation, growth and survival. E6 effects this oncogenic phenotype in part through inhibitory protein-protein interactions (PPIs) and accelerated degradation of proteins with tumor suppressor properties, such as p53 and caspase 8. Interfering with the binding between E6 and its cellular partners may therefore represent a reasonable pharmacological intervention in HPV+ tumors. In this study, we probed a small-molecule library using AlphaScreen™ technology to discover novel E6 inhibitors. Following a cascade of screens we identified and prioritized one hit compound. Structure activity relationship (SAR) studies of this lead uncovered an analog, 30-hydroxygambogic acid (GA-OH), that displayed improved activity. Further testing of this analog in a panel of HPV+ and HPV- cell lines showed good potency and a large window of selectivity as demonstrated by apoptosis induction and significant inhibition of cell growth, cell survival in HPV+ cells. In summary, GA-OH may serve as a starting point for the development of potent E6-specific inhibitors.

Oxidative stress markers in patient-derived non-cancerous cervical tissues and cells.

High-risk human papillomaviruses (HPV) are the causative agents of cervical cancer. However, not all infected women develop cervical cancer. Cervical tumorigenesis is characterized by a multifactorial etiology, with oxidative stress (OS) likely playing a major role. In addition to exogenous sources, metabolic processes also contribute to OS. In principle, variability in levels of cervical OS has the potential to influence the likelihood of conversion to cervical cancer. To ask whether such variability indeed existed, we assessed the levels of ROS and the oxidative DNA damage biomarker 8-oxodG in normal non-cancerous cervical tissues and cells obtained from women with uterovaginal pelvic organ prolapse following vaginal hysterectomy. We demonstrated five and ten-fold variability between tissues isolated from the transformation zone (TZ) and ectocervix (EC) of different women, respectively. Despite the greater variability (likely due to differences in tissue composition), the overall pattern of ROS levels in EC tissues mirrored those obtained in their corresponding TZ tissues. Our results also show that the levels of ROS in TZ tissues were always higher than or equal to those found in the respective EC tissues, providing a possible explanation for TZ tissue being the primary target for HPV infection and cervical carcinogenesis. Interestingly, primary keratinocytes isolated and cultured from these cervical specimens also displayed high variability in ROS levels, with some strongly mirroring the levels of ROS observed in their corresponding tissues, while others were less closely associated. Finally, we demonstrated that the levels of DNA damage mirrored the levels of ROS in the cultured primary cells. Understanding the factors and mechanisms that dispose certain individuals to develop cervical cancer has the potential to enable the development of approaches that make the conversion of HPV infection to cancer development even more rare.

Methylomes in Vegans versus Pescatarians and Nonvegetarians.

Epigenetic studies in animal models have demonstrated that diet affects gene regulation by altering methylation patterns. We interrogated methylomes in humans who have different sources of protein in their diet. We compared methylation of DNA isolated from buffy coat in 38 vegans, 41 pescatarians and 68 nonvegetarians. Methylation data were obtained using Infinium HumanMethylation450 arrays and analyzed using the Partek Genomic software. Differences in differentially methylated sites were small, though with the use of relaxed statistical tests we did identify diet-associated differences. To further test the validity of these observations, we performed separate and independent comparisons of the methylation differences between vegans and nonvegetarians, and between vegans and pescatarians. The detected differences were then examined to determine if they were enriched in specific pathways. Pathway analysis revealed enrichment of several specific processes, including homeobox transcription and glutamate transport. The detected differences in DNA methylation patterns between vegans, pescatarians, and nonvegetarians enabled us to identify 77 CpG sites that may be sensitive to diet and/or lifestyle, though high levels of individual-specific differences were also noted.

Chronic oxidative stress increases the integration frequency of foreign DNA and human papillomavirus 16 in human keratinocytes.

Cervical cancer is the second most common cancer, and the fourth most common cause of cancer death in women worldwide. Nearly all of these cases are caused by high-risk HPVs (HR HPVs), of which HPV16 is the most prevalent type. In most cervical cancer specimens, HR HPVs are found integrated into the human genome, indicating that integration is a key event in cervical tumor development. An understanding of the mechanisms that promote integration may therefore represent a unique opportunity to intercept carcinogenesis. To begin identifying these mechanisms, we tested the hypothesis that chronic oxidative stress (OS) induced by virus- and environmentallymediated factors can induce DNA damage, and thereby increase the frequency with which HPV integrates into the host genome. We found that virus-mediated factors are likely involved, as expression of E6*, a splice isoform of HPV16 E6, increased the levels of reactive oxygen species (ROS), caused oxidative DNA damage, and increased the frequency of plasmid DNA integration as assessed by colony formation assays. To assess the influence of environmentally induced chronic OS, we used L-Buthionine-sulfoximine (BSO) to lower the level of the intracellular antioxidant glutathione. Similar to our observations with E6*, glutathione depletion by BSO also increased ROS levels, caused oxidative DNA damage and increased the integration frequency of plasmid DNA. Finally, under conditions of chronic OS, we were able to induce and characterize a few independent events in which episomal HPV16 integrated into the host genome of cervical keratinocytes. Our results support a chain of events leading from induction of oxidative stress, to DNA damage, to viral integration, and ultimately to carcinogenesis.

Linifanib (ABT-869), enhances cytotoxicity with poly (ADP-ribose) polymerase inhibitor, veliparib (ABT-888), in head and neck carcinoma cells.

OBJECTIVES: PARP inhibitors (PARPi) may provide an opportunity to gain selective killing of tumor cells which have deficiencies in cellular DNA repair systems. We previously demonstrated linifanib (ABT-869), a multi-receptor tyrosine kinase inhibitor of VEGF and PDGF receptor families, radiosensitized Head and Neck Squamous Cell Carcinoma cells (HNSCC) via inhibiting STAT3 activation. Given that STAT3 can modulate DNA damage response (DDR) pathway, in this study, we evaluate the effects of linifanib to enhance cytotoxicity with the PARPi, veliparib (ABT-888), in HNSCC. MATERIALS AND METHODS: UMSCC-22A and UMSCC-22B cells were treated with linifanib (ABT-869) and veliparib (ABT-888). Cell viability, cytotoxicity, apoptosis induction, DNA single strand break (SSB) and double strand break (DSB) damages were examined by MTT assay, colony formation assay, flow cytometry and comet assay. In addition, the expression of DNA homologous recombination repair protein Rad51, γH2AX, a double strand break marker and cleaved PARP, an apoptotic cell death marker, were assessed using western immunoblotting. RESULTS: Combination treatment resulted in more cell growth inhibition, induction of apoptosis, DNA damages and double strand breaks, lower expression of Rad51, increase γH2AX expression and PARP cleavage. CONCLUSION: These data suggest the possibility of combining targeted therapeutic such as linifanib with veliparib to augment the inhibition of cell growth and apoptosis via synthetic lethality in HNSCC cells. Thus, it may provide a novel therapeutic strategy and improve efficacy and outcome in HNSCC.

Proteomic analysis of cellular response induced by multi-walled carbon nanotubes exposure in A549 cells.

The wide application of multi-walled carbon nanotubes (MWCNT) has raised serious concerns about their safety on human health and the environment. However, the potential harmful effects of MWCNT remain unclear and contradictory. To clarify the potentially toxic effects of MWCNT and to elucidate the associated underlying mechanisms, the effects of MWCNT on human lung adenocarcinoma A549 cells were examined at both the cellular and the protein level. Cytotoxicity and genotoxicity were examined, followed by a proteomic analysis (2-DE coupled with LC-MS/MS) of the cellular response to MWCNT. Our results demonstrate that MWCNT induces cytotoxicity in A549 cells only at relatively high concentrations and longer exposure time. Within a relatively low dosage range (30 µg/ml) and short time period (24 h), MWCNT treatment does not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, at these low doses and times, MWCNT treatment causes significant changes in protein expression. A total of 106 proteins show altered expression at various time points and dosages, and of these, 52 proteins were further identified by MS. Identified proteins are involved in several cellular processes including proliferation, stress, and cellular skeleton organization. In particular, MWCNT treatment causes increases in actin expression. This increase has the potential to contribute to increased migration capacity and may be mediated by reactive oxygen species (ROS).

Quantum dot-related genotoxicity perturbation can be attenuated by PEG encapsulation.

Nanomaterial-biosystem interaction is emerging as a major concern hindering wide adoption of nanomaterials. Using quantum dots (Qdots) of different sizes (Qdot-440nm and Qdot-680nm) as a model system, we studied the effects of polyethylene glycol (PEG) thin-layer surface modification in attenuating Qdot-related cytotoxicity, genotoxicity perturbation and oxidative stress in a cellular system. We found that uncoated Qdots (U-Qdots) made of core/shell CdSe/ZnS could indeed induce cytotoxic effects, including the inhibition of cell growth. Also, both the neutral comet assay and γH2AX foci formation showed that U-Qdots caused significant DNA damage in a time- and dose-dependent manner. In contrast, results from cytotoxicity analysis and γH2AX generation indicate minimal impact on cells after exposure to PEG-coated Qdots. This lack of observed toxic effects from PEG-coated Qdots may be due to the fact that PEG-coating can inhibit ROS generation induced by U-Qdots. Based on these observations, we conclude that the genotoxicity of Qdots could be significantly decreased following proper surface modification, such as PEG encapsulation. In addition, PEG encapsulation may also serve as a general method to attenuate nanotoxicity for other nanoparticles.

Effects of benzo(a)pyrene on the contractile function of the thoracic aorta of Sprague-Dawley rats.

OBJECTIVE: To evaluate the possible vascular effects of an environment carcinogen benzo(a)pyrene (BaP). METHODS: The cytotoxicit of BaP and rat liver S9 (0.25 mg/mL)-activated BaP were examined by MTT assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10(-6) mol/L phenylephrine (PE) in an ex-vivo perfusion system after BaP (100 µmol/L) incubation for 6 h. [Ca(2+)](i) was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks (10 mg/kg, weekly, i.p.). RESULTS: BaP (1-500 µm) did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced ([Ca(2+)](i)) increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCl and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively. CONCLUSION: These results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.

Interleukin 24: mechanisms and therapeutic potential of an anti-cancer gene.

Interleukin 24 (mda-7/IL-24) has been classified as an anti-cancer gene for its ability to selectively induce cell death in cancer cells while having little to no effect on normal cells. Although the exact mechanisms by which IL-24 functions have not been completely elucidated, several pathways have consistently been identified: endoplasmic reticulum stress, ceramide-mediated events, and the generation of reactive oxygen species. In addition to these mechanistic analyses, significant progress has also been reported regarding the clinical potential of this anti-cancer gene. For example, many groups are utilizing mda-7/IL-24 in combination with other cancer therapies. This review examines the current research and potential future of this important anti-cancer gene.

Cisplatin treatment leads to changes in nuclear protein and microRNA expression.

Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5µM cisplatin for different times (2, 12, and 24h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process.

DNA damage induces down-regulation of UDP-glucose ceramide glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells.

DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs.