Third-Generation Tetracyclines: Current Knowledge and Therapeutic Potential.

Tetracyclines constitute a unique class of antibiotic agents, widely prescribed for both community and hospital infections due to their broad spectrum of activity. Acting by disrupting protein synthesis through tight binding to the 30S ribosomal subunit, their interference is typically reversible, rendering them bacteriostatic in action. Resistance to tetracyclines has primarily been associated with changes in pump efflux or ribosomal protection mechanisms. To address this challenge, tetracycline molecules have been chemically modified, resulting in the development of third-generation tetracyclines. These novel tetracyclines offer significant advantages in treating infections, whether used alone or in combination therapies, especially in hospital settings. Beyond their conventional antimicrobial properties, research has highlighted their potential non-antibiotic properties, including their impact on immunomodulation and malignancy. This review will focus on third-generation tetracyclines, namely tigecycline, eravacycline, and omadacycline. We will delve into their mechanisms of action and resistance, while also evaluating their pros and cons over time. Additionally, we will explore their therapeutic potential, analyzing their primary indications of prescription, potential future uses, and non-antibiotic features. This review aims to provide valuable insights into the clinical applications of third-generation tetracyclines, thereby enhancing understanding and guiding optimal clinical use.

Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production.

Multiple myeloma is an incurable plasma cell malignancy. Most patients end up relapsing and developing resistance to antineoplastic drugs, like bortezomib. Antibiotic tigecycline has activity against myeloma. This study analyzed tigecycline and bortezomib combination on cell lines and plasma cells from myeloma patients. Apoptosis, autophagic vesicles, mitochondrial mass, mitochondrial superoxide, cell cycle, and hydrogen peroxide were studied by flow cytometry. In addition, mitochondrial antioxidants and electron transport chain complexes were quantified by reverse transcription real-time PCR (RT-qPCR) or western blot. Cell metabolism and mitochondrial activity were characterized by Seahorse and RT-qPCR. We found that the addition of tigecycline to bortezomib reduces apoptosis in proportion to tigecycline concentration. Supporting this, the combination of both drugs counteracts bortezomib in vitro individual effects on the cell cycle, reduces autophagy and mitophagy markers, and reverts bortezomib-induced increase in mitochondrial superoxide. Changes in mitochondrial homeostasis and MYC upregulation may account for some of these findings. These data not only advise to avoid considering tigecycline and bortezomib combination for treating myeloma, but caution on the potential adverse impact of treating infections with this antibiotic in myeloma patients under bortezomib treatment.

Regulation of metastatic potential by drug repurposing and mitochondrial targeting in colorectal cancer cells.

BACKGROUND: Increased mitochondrial activities contributing to cancer cell proliferation, invasion, and metastasis have been reported in different cancers; however, studies on the therapeutic targeting of mitochondria in regulating cell proliferation and invasiveness are limited. Because mitochondria are believed to have evolved through bacterial invasion in mammalian cells, antibiotics could provide an alternative approach to target mitochondria, especially in cancers with increased mitochondrial activities. In this study, we investigated the therapeutic potential of bacteriostatic antibiotics in regulating the growth potential of colorectal cancer (CRC) cells, which differ in their metastatic potential and mitochondrial functions. METHODS: A combination of viability, cell migration, and spheroid formation assays was used to measure the effect on metastatic potential. The effect on mitochondrial mechanisms was investigated by measuring mitochondrial DNA copy number by qPCR, biogenesis (by qPCR and immunoblotting), and functions by measuring reactive oxygen species, membrane potential, and ATP using standard methods. In addition, the effect on assembly and activities of respiratory chain (RC) complexes was determined using blue native gel electrophoresis and in-gel assays, respectively). Changes in metastatic and cell death signaling were measured by immunoblotting with specific marker proteins and compared between CRC cells. RESULTS: Both tigecycline and tetracycline effectively reduced the viability, migration, and spheroid-forming capacity of highly metastatic CRC cells. This increased sensitivity was attributed to reduced mtDNA content, mitochondrial biogenesis, ATP content, membrane potential, and increased oxidative stress. Specifically, complex I assembly and activity were significantly inhibited by these antibiotics in high-metastatic cells. Significant down-regulation in the expression of mitochondrial-mediated survival pathways, such as phospho-AKT, cMYC, phospho-SRC, and phospho-FAK, and upregulation in cell death (apoptosis and autophagy) were observed, which contributed to the enhanced sensitivity of highly metastatic CRC cells toward these antibiotics. In addition, the combined treatment of the CRC chemotherapeutic agent oxaliplatin with tigecycline/tetracycline at physiological concentrations effectively sensitized these cells at early time points. CONCLUSION: Altogether, our study reports that bacterial antibiotics, such as tigecycline and tetracycline, target mitochondrial functions specifically mitochondrial complex I architecture and activity and would be useful in combination with cancer chemotherapeutics for high metastatic conditions.

Gramine sensitizes Klebsiella pneumoniae to tigecycline killing.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.

Inhibition of mitochondrial function by approved drugs overcomes nasopharyngeal carcinoma chemoresistance.

The development of chemo-resistance in nasopharyngeal carcinoma (NPC) presents a significant therapeutic challenge, and its underlying mechanisms remain poorly understood. In our previous studies, we highlighted the association between isoprenylcysteine carboxylmethyltransferase (ICMT) and chemoresistance in NPC. In this current research, we revealed that both 5-FU and cisplatin-resistant NPC cells exhibited elevated mitochondrial function and increased expression of mitochondrial genes, independent of ICMT. Our investigations further showed that classic mitochondrial inhibitors, such as oligomycin, antimycin, and rotenone, were notably more effective in reducing viability in chemo-resistant NPC cells compared to parental cells. Moreover, we identified two antimicrobial drugs, tigecycline and atovaquone, recognized as mitochondrial inhibitors, as potent agents for decreasing chemo-resistant NPC cells by targeting mitochondrial respiration. Remarkably, tigecycline and atovaquone, administered at tolerable doses, inhibited chemo-resistant NPC growth in mouse models and extended overall survival rates. This work unveils the efficacy of mitochondrial inhibition as a promising strategy to overcome chemo-resistance in NPC. Additionally, our findings highlight the potential repurposing of clinically available drugs like tigecycline and atovaquone for treating NPC patients who develop chemoresistance.

Tigecycline causes loss of cell viability mediated by mitochondrial OXPHOS and RAC1 in hepatocellular carcinoma cells.

BACKGROUND: Despite recent advances in locoregional, systemic, and novel checkpoint inhibitor treatment, hepatocellular carcinoma (HCC) is still associated with poor prognosis. The feasibility of potentially curative liver resection (LR) and transplantation (LT) is limited by the underlying liver disease and a shortage of organ donors. Especially after LR, high recurrence rates present a problem and circulating tumor cells are a major cause of extrahepatic recurrence. Tigecycline, a commonly used glycylcycline antibiotic, has been shown to have antitumorigenic effects and could be used as a perioperative and adjuvant therapeutic strategy to target circulating tumor cells. We aimed to investigate the effect of tigecycline on HCC cell lines and its mechanisms of action. METHODS: Huh7, HepG2, Hep3B, and immortalized hepatocytes underwent incubation with clinically relevant tigecycline concentrations, and the influence on proliferation, migration, and invasion was assessed in two- and three-dimensional in vitro assays, respectively. Bioinformatic analysis was used to identify specific targets of tigecycline. The expression of RAC1 was detected using western blot, RT-PCR and RNA sequencing. ELISA and flow cytometry were utilized to measure reactive oxygen species (ROS) generation upon tigecycline treatment and flow cytometry to detect alterations in cell cycle. Changes in mitochondrial function were detected via seahorse analysis. RNA sequencing was performed to examine involved pathways. RESULTS: Tigecycline treatment resulted in a significant reduction of mitochondrial function with concomitantly preserved mitochondrial size, which preceded the observed decrease in HCC cell viability. The sensitivity of HCC cells to tigecycline treatment was higher than that of immortalized non-cancerous THLE-2 hepatocytes. Tigecycline inhibited both migratory and invasive properties. Tigecycline application led to an increase of detected ROS and an S-phase cell cycle arrest. Bioinformatic analysis identified RAC1 as a likely target for tigecycline and the expression of this molecule was increased in HCC cells as a result of tigecycline treatment. CONCLUSION: Our study provides evidence for the antiproliferative effect of tigecycline in HCC. We show for the first time that this effect, likely to be mediated by reduced mitochondrial function, is associated with increased expression of RAC1. The reported effects of tigecycline with clinically relevant and achievable doses on HCC cells lay the groundwork for a conceivable use of this agent in cancer treatment.

Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a highly lethal hematological malignancy in adults and children. Abnormal proliferation of leukemia stem cells (LSC) with CD34+ and CD38- phenotypes are the main clinical features of AML. Patients with AML face drug resistance and treatment failure due to a default in stem and progenitor cells. Therefore, defining LSC properties is necessary for targeting leukemia-initiating cells. Mitochondrial mass and activity increase in AML initiating cells compared with normal stem cells. This idea has offered the inhibition of the mitochondrial translation machinery to reduce the number of leukemia-initiating cells in patients with AML Tigecycline is an FDA-approved microbial antibiotic that inhibits oxidative phosphorylation in mitochondria, resulting in the suppression of leukemia cell proliferation with little toxicity to normal cells. Thus, the present study was conducted to evaluate whether LSC is influenced by mitochondrial inhibition. We measured the IC50 of tigecycline in KG-1a AML cell lines. KG-1a AML cell lines were separated into CD34+ and CD34- cells by MACS. In the following, these cells were treated with 20 µM (IC50) tigecycline. The expression of Annexin/PI, Caspase 3, apoptotic genes (BCL2, BCLX, BAX, BAD, and P53) and proteins (P53, BAX, BCL2 and Caspase 9) was evaluated in CD34+ , CD34- and KG-1a AML cells. In addition, the telomere length and expression of hTERT were evaluated in this study. The results indicated that BCl2 (gene and protein) and BCLX gene dramatically decreased. In addition, BAD, BAX, and P53 gene and protein expression significantly increased in CD34+ AML cells compared to CD34- AML cells. The results also suggested that tigecycline induced intrinsic (Cleaved-caspase 9/Pro-Caspase 9 ratio) and p53-mediated apoptosis. Furthermore, hTERT gene expression and telomere length decreased in the tigecycline-treated groups. Taken together, our findings indicate that inhibition of mitochondrial activity with tigecycline can induce apoptosis in cancer stem cells and can be used as a novel method for cancer therapy.

The effect of mitochondria inhibition on natural killer cells cytotoxicity in triple-negative breast cancer cells.

Triple-Negative Breast Cancer (TNBC), the most common invasive breast cancer, depicts cancer poor response to conventional therapies. The clinical management of TNBC is a challenging issue. Natural killer (NK) cell therapy in the field of cancer treatment is rapidly growing however, regarding the immunogenicity of breast cancer cells, this type of therapy has shown limited efficacy. Recently, targeting tumor biomarkers has revolutionized the field of cancer therapy. Mitochondria affects apoptosis and innate immunity. Therefore, in this study, mitochondria were inhibited with Tigecycline in stimulating the cytotoxicity of NK cells against TNBC cell lines. MDA-MB-468 and MDA-MB-231 were cultured and treated with IC50 (the half-maximal inhibitory concentration) level of Tigecycline for 48 h and afterward co-cultured with peripheral blood NK cells for 5 h. Lastly, the inhibitory effects of mitochondria on the cytotoxicity of NK cells and apoptosis of TNBC cells were evaluated. Moreover, the expression of apoptotic-related genes was studied. The results showed that mitochondria inhibition increased NK cells cytotoxicity against TNBC cells. Moreover, NK cell/mitochondria inhibition in a combinative form improved apoptosis in TNBC cells by the upregulation of Bad and Bid expression. In conclusion, Tigecycline inhibited mitochondria and sensitized TNBC cells to NK cell therapy. Therefore, mitochondria inhibition could help NK cells function properly.

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OBJECTIVES: Gram-positive cocci is the main pathogen responsible for early infection after liver transplantation (LT), posing a huge threat to the prognosis of liver transplant recipients. This study aims to analyze the distribution and drug resistance of Gram-positive cocci, the risk factors for infections and efficacy of antibiotics within 2 months after LT, and to guide the prevention and treatment of these infections. METHODS: In this study, data of pathogenic bacteria distribution, drug resistance and therapeutic efficacy were collected from 39 Gram-positive cocci infections among 256 patients who received liver transplantation from donation after citizens' death in the Third Xiangya Hospital of Central South University from January 2019 to July 2022, and risk factors for Gram-positive cocci infection were analyzed. RESULTS: Enterococcus faecium was the dominant pathogenic bacteria (33/51, 64.7%), followed by Enterococcus faecalis (11/51, 21.6%). The most common sites of infection were abdominal cavity/biliary tract (13/256, 5.1%) and urinary tract (10/256, 3.9%). Fifty (98%) of the 51 Gram-positive cocci infections occurred within 1 month after LT. The most sensitive drugs to Gram-positive cocci were teicoplanin, tigecycline, linezolid and vancomycin. Vancomycin was not used in all patients, considering its nephrotoxicity. Vancomycin was not administered to all patients in view of its nephrotoxicity.There was no significant difference between the efficacy of daptomycin and teicoplanin in the prevention of cocci infection (P>0.05). Univariate analysis indicated that preoperative Model for End-Stage Liver Disease (MELD) score >25 (P=0.005), intraoperative red blood cell infusion ≥12 U (P=0.013) and exposure to more than 2 intravenous antibiotics post-LT (P=0.003) were related to Gram-positive cocci infections. Multivariate logistic regression analysis revealed that preoperative MELD score >25 (OR=2.378, 95% CI 1.124 to 5.032, P=0.024) and intraoperative red blood cell transfusion ≥ 12 U (OR=2.757, 95% CI 1.227 to 6.195, P=0.014) were independent risk factors for Gram-positive cocci infections after LT. Postoperative Gram-positive cocci infections were reduced in LT recipients exposing to more than two intravenous antibiotics post-LT (OR=0.269, 95% CI 0.121 to 0.598, P=0.001). CONCLUSIONS: Gram-positive cocci infections occurring early after liver transplantation were dominated by Enterococcus faecalis infections at the abdominal/biliary tract and urinary tract. Teicoplanin, tigecycline and linezolid were anti-cocci sensitive drugs. Daptomycin and teicoplanin were equally effective in preventing cocci infections due to Gram-positive cocci. Patients with high preoperative MELD score and massive intraoperative red blood cell transfusion were more likely to suffer Gram-positive cocci infection after surgery. Postoperative Gram-positive cocci infections were reduced in recipients exposing to more than two intravenous antibiotics post-LT.

Multiple heteroresistance to tigecycline and colistin in Acinetobacter baumannii isolates and its implications for combined antibiotic treatment.

BACKGROUND: We investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations. METHODS: We performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays. RESULTS: All tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells. CONCLUSION: Our results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections.

The Assessment of Anti-Melanoma Potential of Tigecycline-Cellular and Molecular Studies of Cell Proliferation, Apoptosis and Autophagy on Amelanotic and Melanotic Melanoma Cells.

High mortality, aggressiveness, and the relatively low effectiveness of therapy make melanoma the most dangerous of skin cancers. Previously published studies presented the promising therapeutic potential of minocycline, doxycycline, and chlortetracycline on melanoma cells. This study aimed to assess the cytotoxicity of tigecycline, a third-generation tetracycline, on melanotic (COLO 829) and amelanotic (A375) melanoma cell lines. The obtained results showed that tigecycline, proportionally to the concentration and incubation time, efficiently inhibited proliferation of both types of melanoma cells. The effect was accompanied by the dysregulation of the cell cycle, the depolarization of the mitochondrial membrane, and a decrease in the reduced thiols and the levels of MITF and p44/42 MAPK. However, the ability to induce apoptosis was only found in COLO 829 melanoma cells. A375 cells appeared to be more resistant to the treatment with tigecycline. The drug did not induce apoptosis but caused an increase in LC3A/B protein levels-an autophagy marker. The observed differences in drug action on the tested cell lines also involved an increase in p21 and p16 protein levels in melanotic melanoma, which was related to cell cycle arrest in the G1/G0 phase. The greater sensitivity of melanotic melanoma cells to the action of tigecycline suggests the possibility of considering the use of the drug in targeted therapy.

The Investigation on Nosocomial Infection of Acinetobacter baumannii and the Clinical Analysis of Sequential Therapy of Cefoperazone/Sulbactam Sodium for Intracranial Infection.

Background: Intracranial infection is a serious complication after neurosurgery. According to a survey, the incidence of intracranial infection is about 2.2%-2.6%, and patients with severe symptoms may even pose a threat to their life safety. Objective: To explore the risk factors for intracranial infection caused by Acinetobacter baumannii after surgery and the clinical effect of sequential therapy of cefoperazone/sulbactam sodium. Methods: In this study, a retrospective study was used. In this case-control study, 48 cases of intracranial Acinetobacter baumannii infection after neurosurgery in our hospital from January 2016 to December 2021 were selected as the infection group, and 96 patients without intracranial infection after surgery during the same period were selected as the control group to study all kinds of related factors and analyze the risk factors for intracranial Acinetobacter baumannii infection; in addition, in accordance with the therapeutic regimen for anti-infection, the infection group was divided into the tigecycline group (patients with tigecycline therapy in this group) and the combined group (patients with tigecycline combined with cefoperazone/sulbactam sequential therapy), with 24 cases in each group in order to compare the therapeutic effects of the two groups. Results: Logistic regression factor model results show that increasing age of patients, surgical treatment for intracranial tumor or craniocerebral trauma, postoperative drainage time (≥3 days), and postoperative hospital stay (≥10 days) were the risk factors for postoperative intracranial infection of Acinetobacter baumannii in neurosurgical patients (P < 0.05), and postoperative prophylactic antibiotic treatment can reduce the incidence of intracranial infection (P < 0.05). The cerebrospinal fluid nucleated cell count, serum CRP, and serum PCT in the combined group 72 h after treatment were lower than those in the tigecycline group, and the difference was statistically significant (P < 0.05). Compared with the clinical efficacy after 72-hour treatment, the cure rate and effective rate in the combined treatment group were 83.33% and 16.67%, respectively, and those in the tigecycline group were 54.17% and 33.33%, respectively. The invalid interest rate was 12.50%, and the combined treatment group was superior to the tigecycline group (P < 0.05). Conclusion: For patients with craniocerebral surgery, targeted preventive interventions should be carried out for the risk factors that may lead to intracranial Acinetobacter baumannii infection. The clinical effect of tigecycline combined with cefoperazone and sulbactam sodium sequentially in the treatment of intracranial Acinetobacter baumannii infection is better.

In vitro activity of tigecycline and proteomic analysis of tigecycline adaptation strategies in clinical Enterococcus faecalis isolates from China.

OBJECTIVES: This study aimed to investigate the in vitro activities of tigecycline (TGC) and the underlying molecular mechanisms of TGC stress response and resistance in clinical Enterococcus faecalis isolates from China. METHODS: Antimicrobial susceptibility and antibiofilm activities of TGC in 399 E. faecalis isolates were evaluated. Heteroresistance was evaluated by population analysis profiling. Resistance and heteroresistance mechanisms were investigated by identifying genetic mutations in tetracycline (tet) target sites and through analysis of efflux protein inhibitors (EPIs). Furthermore, quantitative proteomics was used to investigate the global proteomic response of E. faecalis to TGC stress, as well as the resistance mechanisms of TGC within in vitro induced resistant isolate. RESULTS: TGC minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates were ≤0.5 mg/L. TGC displayed remarkable inhibitory activity against biofilm formation. The occurrence rate of TGC heteroresistance was 1.75% (7/399), and the increased TGC MIC values of heteroresistance-derived clones could be reversed by EPI. TGC resistance was associated with mutations in the 16S rRNA site or 30S ribosomal protein S10. A total of 105 and 356 differentially expressed proteins was identified after being exposed to 1/2× MIC concentrations of TGC, while 356 differentially expressed proteins was identified in TGC-resistant isolate. The differentially expressed proteins were enriched in the translation and DNA replication process. In addition, multiple adenosine triphosphate (ATP)-binding cassette (ABC) transporters were upregulated. CONCLUSIONS: TGC exhibited excellent activity against a substantial proportion of clinical isolates from China. However, E. faecalis exhibited a strong adaptation mechanism during TGC exposure: mutation of TGC target sites and elevated expression of efflux pumps under TGC selection, resulting in TGC resistance.

[Anti-tumor activity of tigecycline: a review].

Tigecycline is a novel glycylcycline antibacterial drug, which shows both antibiotic function and anti-tumor activity. This review summarizes the single and combined use of tigecycline for tumor treatment and the underpinning mechanisms. As an inhibitor for mitochondrial DNA translation, tigecycline affects the proliferation, migration, and invasion of tumor cells mainly through inhibiting mitochondrial protein synthesis and inducing mitochondrial dysfunction. Although the effect of tigecycline monotherapy is controversial, the efficacy of combined use of tigecycline is satisfactory. Therefore, it is important to explore the molecular mechanisms underpinning the anti-tumor activity of tigecycline, with the aim to use it as a cheap and effective new anti-tumor drug.

Combined effect of Polymyxin B and Tigecycline to overcome Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae.

We assessed the prevalence of polymyxin B (PMB)- and tigecycline (TGC)-heteroresistant Klebsiella pneumoniae isolates and investigated the combined effect of PMB and TGC against dual-heteroresistant K. pneumoniae. Ninety-five nonduplicated carbapenem-resistant K. pneumoniae (CRKP) clinical isolates were collected from a tertiary-care teaching hospital in China. PCR was used to detect the resistant genes among the CRKP isolates. Population analysis profiling (PAP) was carried out to evaluate the existence of heteroresistance. A time-kill assay of PMB combined with TGC was conducted against heteroresistant K. pneumoniae strains. Real-time PCR was performed to determine the pmrA, phoP, and acrB expression levels. Among them, 74 isolates (77.9%) were susceptible to TGC, and 90 isolates (94.7%) were susceptible to PMB. In addition, of the TGC-susceptible isolates, 49 strains (66.2%) exhibited heteroresistant phenotypes. All of the PMB-susceptible isolates showed heteroresistant phenotypes. Forty-six isolates (48.4%) were heteroresistant to both TGC and PMB. All of the isolates carried the blaKPC gene, and one strain carried both blaKPC and blaNDM genes. The time-kill assay revealed in four isolates that early bactericidal activity could be triggered by the combination of PMB and TGC, and there was no regrowth, even at a relatively lower concentration (0.125 mg/liter PMB with 1 mg/liter TGC). Upregulated expression of pmrA, phoP, and acrB indicated that heteroresistance could be related to two-component systems and the AcrAB-TolC efflux pump. The combination of PMB and TGC may be a treatment strategy for those infected with CRKP heteroresistant to PMB and/or TGC. IMPORTANCE Tigecycline and colistin are two of the last treatment options remaining for carbapenem-resistant Enterobacteriaceae. Unfortunately, tigecycline resistance and colistin heteroresistance are also increasing rapidly. In the current study, we identified a high prevalence of heteroresistance to both PMB and TGC among clinical isolates of carbapenem-resistant K. pneumoniae (CRKP). The resistant subpopulations could survive pressure from TGC or PMB but were killed by the combination at a relatively low dose. It is proposed that the combination of PMB and TGC may be a treatment strategy for patients who are infected with CRKP heteroresistant to PMB or TGC.

Activation of the integrated stress response confers vulnerability to mitoribosome-targeting antibiotics in melanoma.

The ability to adapt to environmental stress, including therapeutic insult, contributes to tumor evolution and drug resistance. In suboptimal conditions, the integrated stress response (ISR) promotes survival by dampening cytosolic translation. We show that ISR-dependent survival also relies on a concomitant up-regulation of mitochondrial protein synthesis, a vulnerability that can be exploited using mitoribosome-targeting antibiotics. Accordingly, such agents sensitized to MAPK inhibition, thus preventing the development of resistance in BRAFV600E melanoma models. Additionally, this treatment compromised the growth of melanomas that exhibited elevated ISR activity and resistance to both immunotherapy and targeted therapy. In keeping with this, pharmacological inactivation of ISR, or silencing of ATF4, rescued the antitumoral response to the tetracyclines. Moreover, a melanoma patient exposed to doxycycline experienced complete and long-lasting response of a treatment-resistant lesion. Our study indicates that the repurposing of mitoribosome-targeting antibiotics offers a rational salvage strategy for targeted therapy in BRAF mutant melanoma and a therapeutic option for NRAS-driven and immunotherapy-resistant tumors.

A systematic review of implications, mechanisms, and stability of in vivo emergent resistance to colistin and tigecycline in Acinetobacter baumannii.

The potential of A. baumannii for acquired resistance to last resort antibiotics (colistin and tigecycline) during treatment has important clinical implications, especially when dealing with patients failing to improve despite treatment with an active antimicrobial. However, the relevant literature remains scattered. Therefore, a systematic search was conducted in PubMed and Scopus. Several studies reported emergence of resistance to colistin or tigecycline during treatment, in most cases (86%) resulting in persistent or recurrent infections, especially in cases of emergent resistance without fitness cost. Lipopolysaccharide modification in the case of colistin and overexpression of efflux pumps in the case of tigecycline were the main mechanisms of resistance. Emergent colistin resistance is often associated with fitness cost which may result in re-emergence of the fitter and more virulent colistin susceptible strain after cessation of antibiotic pressure. Prospective studies are needed to determine the frequency of emergent resistance during treatment and its impact on patient outcomes.

ETNK1 mutations induce a mutator phenotype that can be reverted with phosphoethanolamine.

Recurrent somatic mutations in ETNK1 (Ethanolamine-Kinase-1) were identified in several myeloid malignancies and are responsible for a reduced enzymatic activity. Here, we demonstrate in primary leukemic cells and in cell lines that mutated ETNK1 causes a significant increase in mitochondrial activity, ROS production, and Histone H2AX phosphorylation, ultimately driving the increased accumulation of new mutations. We also show that phosphoethanolamine, the metabolic product of ETNK1, negatively controls mitochondrial activity through a direct competition with succinate at mitochondrial complex II. Hence, reduced intracellular phosphoethanolamine causes mitochondria hyperactivation, ROS production, and DNA damage. Treatment with phosphoethanolamine is able to counteract complex II hyperactivation and to restore a normal phenotype.

Carbon ion combined with tigecycline inhibits lung cancer cell proliferation by inducing mitochondrial dysfunction.

AIMS: Mitochondrial dysfunction is receiving considerable attention due to irreplaceable biological function of mitochondria. Ionizing radiation and tigecycline (TIG) alone can cause mitochondrial dysfunction, playing important role in tumor therapy. However, prior studies fail to investigate combined mechanism of carbon ion irradiation (IR) and TIG on tumor proliferation inhibition. The study aimed to explore the combined effects of both on autophagy and apoptosis. MATERIALS AND METHODS: NSCLC cells A549 and H1299 were treated with carbon ion, TIG, or both. Cell survival rate, autophagy, apoptosis, expression of mitochondrial signaling proteins were determined by clone formation assay, immunofluorescence of LC3B, flow cytometry and western blotting, respectively; ATP content, mitochondrial membrane potential (MMP) and Ca2+ level in mitochondria were used to assessed mitochondrial function. KEY FINDINGS: Results showed IR combined TIG inhibited cells proliferation by increasing apoptosis in both cells and enhancing autophagy in H1299 cells. Additionally, combination treatment induced the most severe mitochondrial dysfunction by sharply reducing ATP, MMP and increasing Ca2+ level of mitochondria. Up-regulation and down-regulation of mitochondrial translation proteins (EF-Tu, GFM1 and MRPS12) expression affected apoptosis and autophagy, while the level of p-mTOR was consistent with their expression in both cell types. In A549 cells, p-AMPK level decreased while p-Akt and p-mTOR increased after combination treatment. SIGNIFICANCE: Overall, our results showed that p-Akt and p-AMPK antagonistically targeted p-mTOR to regulate mitochondrial translation proteins to affect autophagy and apoptosis. Furthermore, this study suggests that combination of carbon ion and TIG is a potential therapeutic option against tumors.

Metabolic implication of tigecycline as an efficacious second-line treatment for sorafenib-resistant hepatocellular carcinoma.

Sorafenib represents the current standard of care for patients with advanced-stage hepatocellular carcinoma (HCC). However, acquired drug resistance occurs frequently during therapy and is accompanied by rapid tumor regrowth after sorafenib therapy termination. To identify the mechanism of this therapy-limiting growth resumption, we established robust sorafenib resistance HCC cell models that exhibited mitochondrial dysfunction and chemotherapeutic crossresistance. We found a rapid relapse of tumor cell proliferation after sorafenib withdrawal, which was caused by renewal of mitochondrial structures alongside a metabolic switch toward high electron transport system (ETS) activity. The translation-inhibiting antibiotic tigecycline impaired the biogenesis of mitochondrial DNA-encoded ETS subunits and limited the electron acceptor turnover required for glutamine oxidation. Thereby, tigecycline prevented the tumor relapse in vitro and in murine xenografts in vivo. These results offer a promising second-line therapeutic approach for advanced-stage HCC patients with progressive disease undergoing sorafenib therapy or treatment interruption due to severe adverse events.