VgrG Spike Dictates PAAR Requirement for the Assembly of the Type VI Secretion System.

Widely employed by Gram-negative pathogens for competition and pathogenesis, the type six protein secretion system (T6SS) can inject toxic effectors into neighboring cells through the penetration of a spear-like structure comprising a long Hcp tube and a VgrG-PAAR spike complex. The cone-shaped PAAR is believed to sharpen the T6SS spear for penetration but it remains unclear why PAAR is required for T6SS functions in some bacteria but dispensable in others. Here, we report the conditional requirement of PAAR for T6SS functions in Aeromonas dhakensis, an emerging human pathogen that may cause severe bacteremia. By deleting the two PAAR paralogs, we show that PAAR is not required for T6SS secretion, bacterial killing, or specific effector delivery in A. dhakensis. By constructing combinatorial PAAR and vgrG deletions, we demonstrate that deletion of individual PAAR moderately reduced T6SS functions but double or triple deletions of PAAR in the vgrG deletion mutants severely impaired T6SS functions. Notably, the auxiliary-cluster-encoded PAAR2 and VgrG3 are less critical than the main-cluster-encoded PAAR1 and VgrG1&2 proteins to T6SS functions. In addition, PAAR1 but not PAAR2 contributes to antieukaryotic virulence in amoeba. Our data suggest that, for a multi-PAAR T6SS, the variable role of PAAR paralogs correlates with the VgrG-spike composition that collectively dictates T6SS assembly. IMPORTANCE Gram-negative bacteria often encode multiple paralogs of the cone-shaped PAAR that sits atop the VgrG-spike and is thought to sharpen the spear-like T6SS puncturing device. However, it is unclear why PAAR is required for the assembly of some but not all T6SSs and why there are multiple PAARs if they are not required. Our data delineate a VgrG-mediated conditional requirement for PAAR and suggest a core-auxiliary relationship among different PAAR-VgrG modules that may have been acquired sequentially by the T6SS during evolution.

VgrG-dependent effectors and chaperones modulate the assembly of the type VI secretion system.

The type VI secretion system (T6SS) is a spear-like nanomachine found in gram-negative pathogens for delivery of toxic effectors to neighboring bacterial and host cells. Its assembly requires a tip spike complex consisting of a VgrG-trimer, a PAAR protein, and the interacting effectors. However, how the spike controls T6SS assembly remains elusive. Here we investigated the role of three VgrG-effector pairs in Aeromonas dhakensis strain SSU, a clinical isolate with a constitutively active T6SS. By swapping VgrG tail sequences, we demonstrate that the C-terminal ~30 amino-acid tail dictates effector specificity. Double deletion of vgrG1&2 genes (VgrG3+) abolished T6SS secretion, which can be rescued by ectopically expressing chimeric VgrG3 with a VgrG1/2-tail but not the wild type VgrG3. In addition, deletion of effector-specific chaperones also severely impaired T6SS secretion, despite the presence of intact VgrG and effector proteins, in both SSU and Vibrio cholerae V52. We further show that SSU could deliver a V. cholerae effector VasX when expressing a plasmid-borne chimeric VgrG with VasX-specific VgrG tail and chaperone sequences. Pull-down analyses show that two SSU effectors, TseP and TseC, could interact with their cognate VgrGs, the baseplate protein TssK, and the key assembly chaperone TssA. Effectors TseL and VasX could interact with TssF, TssK and TssA in V. cholerae. Collectively, we demonstrate that chimeric VgrG-effector pairs could bypass the requirement of heterologous VgrG complex and propose that effector-stuffing inside the baseplate complex, facilitated by chaperones and the interaction with structural proteins, serves as a crucial structural determinant for T6SS assembly.

Heterologous Assembly of the Type VI Secretion System Empowers Laboratory Escherichia coli with Antimicrobial and Cell Penetration Capabilities.

The synthetic biology toolbox has amassed a vast number of diverse functional modules, but protein translocation modules for cell penetration and cytosol-to-cytosol delivery remain relatively scarce. The type VI secretion system (T6SS), commonly found in many Gram-negative pathogens, functions as a contractile device to translocate protein toxins to prokaryotic and eukaryotic cells. Here, we have assembled the T6SS of Aeromonas dhakensis, an opportunistic waterborne pathogen, in the common laboratory strain Escherichia coli BL21(DE3). We constructed a series of plasmids (pT6S) carrying the T6SS structural and effector genes under native or tetracycline-inducible promoters, the latter for controlled expression. Using fluorescence microscopy and biochemical analyses, we demonstrate a functional T6SS in E. coli capable of secreting proteins directly into the cytosol of neighboring bacteria and outcompeting a number of drug-resistant pathogens. The heterologous assembly of T6SS not only confers the lab workhorse E. coli with the cytosol-to-cytosol protein delivery capability but also demonstrates the potential for harnessing the T6SS of various pathogens for general protein delivery and antibacterial applications. IMPORTANCE The T6SS is a powerful and versatile protein delivery system. However, the complexity of its macromolecular structure and gene regulation makes it not a trivial task to reconstitute the T6SSs of pathogens in a nonpathogenic host. In this study, we have assembled an inducible T6SS in E. coli BL21(DE3) and demonstrated its functions in protein delivery and antimicrobial activities. The engineered T6SS empowers E. coli to deliver protein cargos into a wide range of prokaryotic and eukaryotic cells.

Development and Validation of Noninvasive MRI-Based Signature for Preoperative Prediction of Early Recurrence in Perihilar Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is a type of hepatobiliary tumor. For perihilar cholangiocarcinoma (pCCA), patients who experience early recurrence (ER) have a poor prognosis. Preoperative accurate prediction of postoperative ER can avoid unnecessary operation; however, prediction is challenging. PURPOSE: To develop a novel signature based on clinical and/or MRI radiomics features of pCCA to preoperatively predict ER. STUDY TYPE: Retrospective. POPULATION: One hundred eighty-four patients (median age, 61.0 years; interquartile range: 53.0-66.8 years) including 115 men and 69 women. FIELD STRENGTH/SEQUENCE: A 1.5 T; volumetric interpolated breath-hold examination (VIBE) sequence. ASSESSMENT: The models were developed from the training set (128 patients) and validated in a separate testing set (56 patients). The contrast-enhanced arterial and portal vein phase MR images of hepatobiliary system were used for extracting radiomics features. The correlation analysis, least absolute shrinkage and selection operator (LASSO) logistic regression (LR), backward stepwise LR were mainly used for radiomics feature selection and modeling (Modelradiomic ). The univariate and multivariate backward stepwise LR were used for preoperative clinical predictors selection and modeling (Modelclinic ). The radiomics and preoperative clinical predictors were combined by multivariate LR method to construct clinic-radiomics nomogram (Modelcombine ). STATISTICAL TESTS: Chi-squared (χ2 ) test or Fisher's exact test, Mann-Whitney U-test or t-test, Delong test. Two tailed P < 0.05 was considered statistically significant. RESULTS: Based on the comparison of area under the curves (AUC) using Delong test, Modelclinic and Modelcombine had significantly better performance than Modelradiomic and tumor-node-metastasis (TNM) system in training set. In the testing set, both Modelclinic and Modelcombine had significantly better performance than TNM system, whereas only Modelcombine was significantly superior to Modelradiomic . However, the AUC values were not significantly different between Modelclinic and Modelcombine (P = 0.156 for training set and P = 0.439 for testing set). DATA CONCLUSION: A noninvasive model combining the MRI-based radiomics signature and clinical variables is potential to preoperatively predict ER for pCCA. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 4.

Salinomycin acts through reducing AKT-dependent thymidylate synthase expression to enhance erlotinib-induced cytotoxicity in human lung cancer cells.

Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). Salinomycin, a polyether antibiotic, has been promising a novel therapeutic agent for lung cancer, and down-regulated the expression of thymidylate synthase (TS) in NSCLC cell lines. Previous study showed that against EGFR and TS was strongly synergistic cytotoxicity in NSCLC cells. In this study, we showed that erlotinib (1.25-10µM) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung squamous cell carcinoma H1703 and adenocarcinoma H1975 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of erlotinib. A combination of erlotinib and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced protein levels of phospho-AKT(Ser473), phospho-AKT(Thr308), and TS. Overexpression of a constitutive active AKT (AKT-CA) or Flag-TS expression vector reversed the salinomycin and erlotinib-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the erlotinib-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with erlotinib for lung cancer treatment.

17-(Allylamino)-17-Demethoxygeldanamycin Enhances Etoposide-Induced Cytotoxicity via the Downregulation of Xeroderma Pigmentosum Complementation Group C Expression in Human Lung Squamous Cell Carcinoma Cells.

Etoposide (VP16) is a topoisomerase II inhibitor and has been used for the treatment of non-small cell lung cancer (NSCLC). Xeroderma pigmentosum complementation group C (XPC) protein is a DNA damage recognition factor in nucleotide excision repair and involved in regulating NSCLC cell proliferation and viability. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) enhanced etoposide-induced cytotoxicity in NSCLC cells through modulating the XPC expression. We found that etoposide increased XPC expression in an AKT activation manner in 2 squamous cell carcinoma H1703 and H520 cells. Knockdown of XPC using siRNA or inactivation of AKT by pharmacological inhibitor PI3K inhibitor (LY294002) enhanced the cytotoxic effects of etoposide. In contrast, enforced expression of XPC cDNA or AKT-CA (a constitutively active form of AKT) reduced the cytotoxicity and cell growth inhibition of etoposide. Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of etoposide in NSCLC cells, which were associated with the downregulation of XPC expression and inactivation of AKT. Our findings suggested that the Hsp90 inhibition induced XPC downregulation involved in enhancing the etoposide-induced cytotoxicity in H1703 and H520 cells.

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells.

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells.

Delivery of an Rhs-family nuclease effector reveals direct penetration of the gram-positive cell envelope by a type VI secretion system in Acidovorax citrulli.

The type VI secretion system (T6SS) is a double-tubular nanomachine widely found in gram-negative bacteria. Its spear-like Hcp tube is capable of penetrating a neighboring cell for cytosol-to-cytosol protein delivery. However, gram-positive bacteria have been considered impenetrable to such T6SS action. Here we report that the T6SS of a plant pathogen, Acidovorax citrulli (AC), could deliver an Rhs-family nuclease effector RhsB to kill not only gram-negative but also gram-positive bacteria. Using bioinformatic, biochemical, and genetic assays, we systematically identified T6SS-secreted effectors and determined that RhsB is a crucial antibacterial effector. RhsB contains an N-terminal PAAR domain, a middle Rhs domain, and an unknown C-terminal domain. RhsB is subject to self-cleavage at both its N- and C-terminal domains and its secretion requires the upstream-encoded chaperone EagT2 and VgrG3. The toxic C-terminus of RhsB exhibits DNase activities and such toxicity is neutralized by either of the two downstream immunity proteins, RimB1 and RimB2. Deletion of rhsB significantly impairs the ability of killing Bacillus subtilis while ectopic expression of immunity proteins RimB1 or RimB2 confers protection. We demonstrate that the AC T6SS not only can effectively outcompete Escherichia coli and B. subtilis in planta but also is highly potent in killing other bacterial and fungal species. Collectively, these findings highlight the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.

More Than Just a Spearhead: Diverse Functions of PAAR for Assembly and Delivery of Toxins of the Contractile Injection Systems.

The type VI secretion system (T6SS) belongs to the evolutionarily related group of contractile injection systems that employ a contractile outer sheath to inject a rigid spear-like inner tube into target bacterial and eukaryotic cells. The tip of the rigid tube is often decorated by a PAAR-repeat protein as a key structural component. Many members of the PAAR protein family can also have additional and diverse functions by serving as toxins for those with extended domains or as carriers for interacting toxins. A plethora of toxin modules or modules of unknown functions have been bioinformatically predicted to be associated with PAAR either as a fused domain or as an interacting partner, and yet only a small number of PAAR proteins have been studied, highlighting the exciting and dire need for future research to better understand the diverse PAAR-mediated functions.

Inhibition of thymidine phosphorylase expression by Hsp90 inhibitor potentiates the cytotoxic effect of salinomycin in human non-small-cell lung cancer cells.

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells.

Astaxanthin enhances erlotinib-induced cytotoxicity by p38 MAPK mediated xeroderma pigmentosum complementation group C (XPC) down-regulation in human lung cancer cells.

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects that include anti-cancer and anti-inflammatory properties. Xeroderma pigmentosum complementation group C (XPC) protein is an important DNA damage recognition factor in nucleotide excision repair and is involved in regulating non-small cell lung cancer (NSCLC) cell proliferation and viability. Erlotinib (TarcevaR) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated clinical activity in NSCLC cells. However, whether astaxanthin and erlotinib could induce synergistic cytotoxicity in NSCLC cells through modulating XPC expression is unknown. In this study, we found that p38 MAPK activation by astaxanthin decreased XPC expression in two human lung adenocarcinoma A549 and H1975 cells. Inactivation of p38 MAPK by pharmacological inhibitor SB203580 or the specific small interfering RNA (siRNA) rescued the astaxanthin-reduced XPC mRNA and protein levels. Enforced expression of XPC cDNA or inhibiting the p38 MAPK activity reduced the cytotoxicity and cell growth inhibition of astaxanthin. In contrast, knockdown of XPC using siRNA enhanced the cytotoxic effects of astaxanthin. Moreover, astaxanthin synergistically enhanced cytotoxicity and cell growth inhibition of erlotinib in NSCLC cells, which were associated with the down-regulation of XPC expression and activation of p38 MAPK. Our findings suggested that the astaxanthin induced p38 MAPK mediated XPC down-regulation enhanced the erlotinib-induced cytotoxicity in A549 and H1975 cells.

Salinomycin enhances cisplatin-induced cytotoxicity in human lung cancer cells via down-regulation of AKT-dependent thymidylate synthase expression.

Salinomycin, a polyether antibiotic, acts as a highly selective potassium ionophore and has anticancer activity on various cancer cell lines. Cisplatin has been proved as chemotherapy drug for advanced human non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) is a key enzyme in the pyrimidine salvage pathway, and increased expression of TS is thought to be associated with resistance to cisplatin. In this study, we showed that salinomycin (0.5-2µg/mL) treatment down-regulating of TS expression in an AKT inactivation manner in two NSCLC cell lines, human lung adenocarcinoma A549 and squamous cell carcinoma H1703 cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting AKT activity with PI3K inhibitor LY294002 enhanced the cytotoxicity and cell growth inhibition of salinomycin. A combination of cisplatin and salinomycin resulted in synergistic enhancement of cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-AKT, and TS expression. Overexpression of a constitutive active AKT (AKT-CA) expression vector reversed the salinomycin and cisplatin-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in salinomycin and cisplatin cotreated cells. Our findings suggested that the down-regulation of AKT-mediated TS expression by salinomycin enhanced the cisplatin-induced cytotoxicity in NSCLC cells. These results may provide a rationale to combine salinomycin with cisplatin for lung cancer treatment.

Curcumin downregulates p38 MAPK-dependent X-ray repair cross-complement group 1 (XRCC1) expression to enhance cisplatin-induced cytotoxicity in human lung cancer cells.

Cisplatin is a well-studied and widely used chemotherapeutic agent and is effective in the treatment of the advanced human non-small cell lung cancer (NSCLC). Curcumin is a yellow pigment derived from the rhizome of Curcuma longa and has been proved to have antioxidant and antitumor properties. XRCC1 is an important scaffold protein involved in base excision repair and plays an important role in the development of lung cancer. In this study, we characterize the role of curcumin in the cytotoxicity, p38 MAPK activation, and XRCC1 expression affected by cisplatin in NSCLC cells. We show that curcumin enhanced the cytotoxicity induced by cisplatin in two NSCLC cells, A549 and H1703. Treatment with cisplatin alone increased XRCC1 mRNA and protein expression through p38 MAPK activation. Moreover, SB2023580 (p38 inhibitor) decreased the XRCC1 mRNA and protein stability upon cisplatin treatment. Knockdown of XRCC1 in NSCLC cells by transfection of XRCC1 siRNA or inactivation of p38 MAPK resulted in enhancing the cytotoxicity and cell growth inhibition induced by cisplatin. Curcumin inhibited the expression of XRCC1 in cisplatin-exposed NSCLC cells. Furthermore, transfection with constitutive active MKK6 or HA-p38 MAPK vectors rescued the XRCC1 protein level and also the cell survival suppressed by cisplatin and curcumin combination in A549 and H1703 cells. These findings suggested that the downregulation of XRCC1 expression by curcumin can enhance the chemosensitivity of cisplatin in NSCLC cells.

Astaxanthin down-regulates Rad51 expression via inactivation of AKT kinase to enhance mitomycin C-induced cytotoxicity in human non-small cell lung cancer cells.

Astaxanthin has been demonstrated to exhibit a wide range of beneficial effects, including anti-inflammatory and anti-cancer properties. However, the molecular mechanism of astaxanthin-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination, and studies show that chemo-resistant carcinomas exhibit high levels of Rad51 expression. In this study, astaxanthin treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1703. Astaxanthin treatment (2.5-20 µM) decreased Rad51 expression and phospho-AKT(Ser473) protein level in a time and dose-dependent manner. Furthermore, expression of constitutively active AKT (AKT-CA) vector rescued the decreased Rad51 mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with phosphatidylinositol 3-kinase (PI3K) inhibitors (LY294002 or wortmannin) further decreased the Rad51 expression in astaxanthin-exposed A549 and H1703 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA or cotreatment with LY294002 further enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Additionally, mitomycin C (MMC) as an anti-tumor antibiotic is widely used in clinical NSCLC chemotherapy. Combination of MMC and astaxanthin synergistically resulted in cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced phospho-AKT(Ser473) level and Rad51 expression. Overexpression of AKT-CA or Flag-tagged Rad51 reversed the astaxanthin and MMC-induced synergistic cytotoxicity. In contrast, pretreatment with LY294002 further decreased the cell viability in astaxanthin and MMC co-treated cells. In conclusion, astaxanthin enhances MMC-induced cytotoxicity by decreasing Rad51 expression and AKT activation. These findings may provide rationale to combine astaxanthin with MMC for the treatment of NSCLC.

Minocycline enhances mitomycin C-induced cytotoxicity through down-regulating ERK1/2-mediated Rad51 expression in human non-small cell lung cancer cells.

Minocycline is a semisynthetic tetracycline derivative; it has anti-inflammatory and anti-cancer effects distinct from its antimicrobial function. However, the molecular mechanism of minocycline-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells has not been identified. Rad51 plays a central role in homologous recombination and high levels of Rad51 expression are observed in chemo- or radioresistant carcinomas. Our previous studies have shown that the MKK1/2-ERK1/2 signal pathway maintains the expression of Rad51 in NSCLC cells. In this study, minocycline treatment inhibited cell viability and proliferation of two NSCLC cells, A549 and H1975. Treatment with minocycline decreased Rad51 mRNA and protein levels through MKK1/2-ERK1/2 inactivation. Furthermore, expression of constitutively active MKK1 (MKK1-CA) vectors significantly rescued the decreased Rad51 protein and mRNA levels in minocycline-treated NSCLC cells. However, combined treatment with MKK1/2 inhibitor U0126 and minocycline further decreased the Rad51 expression and cell viability of NSCLC cells. Knocking down Rad51 expression by transfection with small interfering RNA of Rad51 enhanced the cytotoxicity and cell growth inhibition of minocycline. Mitomycin C (MMC) is typically used as a first or second line regimen to treat NSCLC. Compared to a single agent alone, MMC combined with minocycline resulted in cytotoxicity and cell growth inhibition synergistically in NSCLC cells, accompanied with reduced activation of phospho-ERK1/2, and reduced Rad51 protein levels. Overexpression of MKK1-CA or Flag-tagged Rad51 could reverse the minocycline and MMC-induced synergistic cytotoxicity. These findings may have implications for the rational design of future drug regimens incorporating minocycline and MMC for the treatment of NSCLC.