Combinatorial Drug Screening Based on Massive 3D Tumor Cultures Using Micropatterned Array Chips.

The establishment and application of a generalizable three-dimensional (3D) tumor device for high-throughput screening plays an important role in drug discovery and cancer therapeutics. In this study, we introduce a facile microplatform for considerable 3D tumor generation and combinatorial drug screening evaluation. High fidelity of chip fabrication was achieved depending on the simple and well-improved microcontact printing. We demonstrated the high stability and repeatability of the established tumor-on-a-chip system for controllable and massive production of 3D tumors with high size uniformity. Importantly, we accomplished the screening-like chemotherapy investigation involving individual and combinatorial drugs and validated the high accessibility and applicability of the system in 3D tumor-based manipulation and analysis on a large scale. This achievement in tumor-on-a-chip has potential applications in plenty of biomedical fields such as tumor biology, pharmacology, and tissue microengineering. It offers an insight into the development of the popularized microplatform with easy-to-fabricate and easy-to-operate properties for cancer exploration and therapy.

Single-cell droplet microfluidics for biomedical applications.

Single-cell manipulation and analysis is critical to the study of many fundamental biological processes and uncovering cellular heterogeneity, and presents the potential for extremely valuable applications in biomedical fields, including neuroscience, regenerative therapy, early diagnosis, and drug screening. The use of microfluidic technologies in single-cell manipulation and analysis is one of the most promising approaches and enables the creation of innovative conditions that are impractical or impossible to achieve using conventional methods. Herein, an overview of the technological development of single-cell droplet microfluidics is presented. The significant advantages of microfluidic droplet technology, the dynamic parameters affecting droplet production, and the geometric structures of microfluidic devices are emphasized. Furthermore, the progress to date in passive and active droplet generation methods based on microfluidics and various microfluidic tools for the production of single-cell droplets and hydrogel microspheres are summarized. Their key features, achievements, and limitations associated with single-cell droplet and hydrogel formation are discussed. The recent popularized applications of single-cell droplet microfluidics in biomedicine involving small-molecule detection, protein analysis, and drug screening and genetic analysis of single cells are explored too. Finally, the challenges that must be overcome to enable future applications in single-cell droplet microfluidics are highlighted.

Large-scale investigation of single cell activities and response dynamics in a microarray chip with a microfluidics-fabricated microporous membrane.

Microengineering technology involving microfabrication, micropatterning and microfluidics enables promising advances in single cell manipulation and analysis. Herein, we describe a parallel, large-scale, and temporal investigation of diverse single cell activities and response dynamics using a facile-assembled microwell array chip with a microfluidics-molded microporous membrane. We demonstrated that the versatility with respect to geometrical homogeneity and diversity of microporous membrane fabrication, as well as the stability, repeatability, and reproducibility rely on the well-improved molding. Serial and practical operations including controllable single cell trapping, array-like culture or chemical stimulation, and temporal monitoring can be smoothly completed in the chip. We confirmed that the microwell array chip allowed an efficient construction of a single cell array. Using the cell array, on-chip detection of single cell behaviours under various culture and drug therapy conditions to explore phenotypic heterogeneity was achieved in massive and dynamic manners. These achievements provide a facile and reliable methodology for fabricating microporous membranes with precise control and for developing universal microplatforms to perform robust manipulation and versatile analysis of single cells. This work also offers an insight into the development of easy to fabricate/use and market-oriented microsystems for single cell research, pharmaceutical development, and high-throughput screening.

Straightforward neuron micropatterning and neuronal network construction on cell-repellent polydimethylsiloxane using microfluidics-guided functionalized Pluronic modification.

Neuronal cell microengineering involving micropatterning and polydimethylsiloxane (PDMS) microfluidics enables promising advances in microscale neuron control. However, a facile methodology for the precise and effective manipulation of neurons on a cell-repellent PDMS substrate remains challenging. Herein, a simple and straightforward strategy for neuronal cell patterning and neuronal network construction on PDMS based on microfluidics-assisted modification of functionalized Pluronic is described. The cell patterning process simply involves a one-step microfluidic modification and routine in vitro culture. It is demonstrated that multiple types of neuronal cell arrangements with various spatial profiles can be conveniently produced using this patterning tool. The precise control of neuronal cells with high patterning fidelity up to single cell resolution, as well as high adhesion and differentiation, is achieved too. Furthermore, neuronal network construction using the respective cell population and single cell patterning prove to be applicable. This achievement provides a convenient and feasible methodology for engineering neuronal cells on PDMS substrates, which will be useful for applications in many neuron-related microscale analytical research fields, including cell engineering, neurobiology, neuropharmacology, and neuronal sensing.

An integrated microfluidic 3D tumor system for parallel and high-throughput chemotherapy evaluation.

The development of a microplatform with multifunctional integration allowing the dynamic and high-throughput exploration of three-dimensional (3D) cultures is promising for biomedical research. Here, we introduce an integrated microfluidic 3D tumor system with pneumatic manipulation and chemical gradient generation to investigate anticancer therapy in a parallel, controllable, dynamic, and high-throughput manner. The stability of the microfluidic system to realize precise and long-term chemical gradient production was developed. Serial manipulations including active cell trapping, array-like tumor self-assembly and formation, reliable gradient generation, parallel multi-concentration drug stimulation, and real-time tumor analysis were achieved in a single microfluidic device. The microfluidic platform was demonstrated to be stable for high-throughput cell trapping and 3D tumor formation with uniform quantities. On-chip analysis of phenotypic tumor responses to diverse chemotherapies with different concentrations can be conducted in this device. The microfluidic advancement holds great potential for applications in the development of high-performance and multi-functional biomimetic tumor systems and in the fields of cancer research and pharmaceutical development.

Large-Scale Antitumor Screening Based on Heterotypic 3D Tumors Using an Integrated Microfluidic Platform.

Chemotherapy screening plays a crucial role in cancer drug discovery and clinical medicine. Although conventional methods have contributed greatly to macromanipulation of cell populations, profounder insights related to the tumor microenvironment require approaches for completing integrated cell-3D tumor micromanipulation, massive tumor simulation and production, and dynamic and high-throughput tumor analysis. In this study, we introduced an integrated microfluidic platform with multiparallel components for heterotypic 3D tumor reconstruction and antitumor screening. Sequential microfluidic manipulations including sample loading, precise localization, 3D tumor formation, chemical stimulation, on-chip analysis, and tumor recovery for off-chip assessment were permitted and experimentally confirmed in the device on the basis of facile and efficient pneumatic control. Heterotypic 3D tumors with tissue-biomimetic phenotypes can be produced in massive and size-uniform manners. Notably, we accomplished a screening-like chemotherapy assessment involving different heterotypic 3D tumors and antitumor drugs and demonstrated the versatility of the platform in large-scale tumor manipulation and analysis. This advancement in microfluidics has potential applications in the fields of oncology, pharmacology, and tissue engineering and provides insight into the construction of high-performance microsystems for drug development and cancer research.

Facile and Rapid Microcontact Printing of Additive-Free Polydimethylsiloxane for Biological Patterning Diversity.

The development and application of micropatterning technology play a promising role in the manipulation of biological substances and the exploration of life sciences at the microscale. However, the universally adaptable micropatterning method with user-friendly properties for acceptance in routine laboratories remains scarce. Herein, a green, facile, and rapid microcontact printing method is reported for upgrading popularization and diversification of biological patterning. The three-step printing can achieve high simplicity and fidelity of additive-free polydimethylsiloxane (PDMS) micropatterning and chip fabrication within 8 min as well as keep their high stability and diversity. A detailed experimental report is provided to support the advanced microcontact printing method. Furthermore, the applications of easy-to-operate PDMS-patterned chips are extensively validated to complete microdroplet array assembly with spatial control, cell pattern formation with high efficiency and geometry customization, and microtissue assembly and biomimetic tumor construction on a large scale. This straightforward method promotes diverse micropatternings with minimal time, effort, and expertise and maximal biocompatibility, which might broaden its applications in interdisciplinary scientific communities. This work also offers an insight into the establishment of popularized and market-oriented microtools for biomedical purposes such as biosensing, organs on a chip, cancer research, and bioscreening.

Novel vacuum UV/ozone/peroxymonosulfate process for efficient degradation of levofloxacin: Performance evaluation and mechanism insight.

Vacuum UV (VUV) irradiation has advantage in coupling oxidants for organics removal because VUV can dissociate water to produce reactive oxygen species (ROS) in situ and decompose oxidants rapidly. In this study, the synergistic activation of peroxymonosulfate (PMS) by VUV and ozone (O3) was explored via developing a novel integrated VUV/O3/PMS process, and the performance and mechanisms of VUV/O3/PMS for levofloxacin (LEV) degradation were investigated systematically. Results indicated that VUV/O3/PMS could effectively degrade LEV, and the degradation rate was 1.67-18.79 times of its sub-processes. Effects of PMS dosage, O3 dosage, solution pH, anions, and natural organic matter on LEV removal by VUV/O3/PMS were also studied. Besides, hydroxyl radical and sulfate radical were main ROS with contributions of 49.7% and 17.4%, respectively. Moreover, the degradation pathways of LEV in VUV/O3/PMS process were speculated based on density functional theory calculation and by-products detection. Furthermore, synergistic reaction mechanisms in VUV/O3/PMS process were proposed. The energy consumption of VUV/O3/PMS decreased by 22.6%- 88.1% compared to its sub-processes. Finally, the integrated VUV/O3/PMS process showed satisfactory results in removing LEV in actual waters, manifesting VUV/O3/PMS had great application potential and feasibility in removing organics in wastewater reuse.

Butylphthalide inhibits ferroptosis and ameliorates cerebral Ischaemia-Reperfusion injury in rats by activating the Nrf2/HO-1 signalling pathway.

This study aims to investigate whether butylphthalide can inhibit ferroptosis and ameliorate cerebral ischaemia-reperfusion (I/R) injury in rats by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) / heme oxygenase-1 (HO-1) signalling pathway, known for its antioxidative and cytoprotective properties. Middle cerebral artery occlusion reperfusion (MCAO/R) rat models were established. Male rats were randomly divided into five groups: a sham-operated group (sham), MCAO/R group, MCAO/R â€‹+ â€‹ML385 (Nrf2-specific inhibitor) group, MCAO/R â€‹+ â€‹NBP (butylphthalide) group and MCAO/R â€‹+ â€‹ML385 â€‹+ â€‹NBP group. The effect of butylphthalide on cerebral I/R injury was evaluated using neurological deficit scores. The expression levels of Nrf2, HO-1, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and transferrin receptor 1 (TfR1) protein were detected using Western blot. Moreover, the expression levels of GPX4, HO-1 and TfR1 mRNA were determined through real-time fluorescence quantitative reverse transcription polymerase chain reaction. The distribution of Nrf2, HO-1, GPX4 and TfR1 was detected using immunohistochemical staining. The levels of iron and related lipid peroxidation indexes, such as reduced glutathione, reactive oxygen species, malondialdehyde and nitric oxide, were measured using a kit. The changes in mitochondria were observed through transmission electron microscopy. Butylphthalide treatment significantly improved neurological dysfunction, reduced cerebral infarction volume and mitigated histopathological damage in MCAO/R rats. It induced the nuclear translocation of Nrf2 and upregulated HO-1 expression, which was attenuated by ML385. Butylphthalide also attenuated lipid peroxidation, iron accumulation and mitochondrial damage induced by MCAO/R. The expression of GPX4, ACSL4 and TfR1 proteins, as well as their mRNA levels, was modulated through butylphthalide treatment, with improvements observed in mitochondrial morphology. Butylphthalide exerts neuroprotective effects by attenuating neurological dysfunction and ferroptosis in MCAO/R rats through the activation of the Nrf2/HO-1 pathway and inhibition of lipid peroxidation and iron accumulation.

Pentoxifylline protects against cerebral ischaemia-reperfusion injury through ferroptosis regulation via the Nrf2/SLC7A11/GPX4 signalling pathway.

OBJECTIVE: To investigate whether pentoxifylline (PTX) attenuates cerebral ischaemia-reperfusion injury (IRI) in rats by inhibiting ferroptosis and to explore the underlying molecular mechanisms. METHODS: Cerebral IRI was induced in male Sprague-Dawley (SD) rats using middle cerebral artery occlusion (MCAO). The effects of PTX on cerebral ischaemia-reperfusion brain samples were detected through neurological deficit score, staining and electron microscopy; levels of ferroptosis biomarkers from brain samples were detected using kits. Additionally, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), transferrin receptor protein 1, divalent metal transporter 1, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were determined by immunohistochemistry, real-time quantitative polymerase chain reaction and western blotting. RESULTS: Pre-treatment with PTX was found to improve neurological function, evidenced by reduced neurological deficit scores, decreased infarct volume and alleviated pathological features post-MCAO. This improvement was accompanied by reduced lipid peroxidation levels and mitigated mitochondrial damage. Notably, PTX's inhibitory effect on ferroptosis was characterised by enhanced Nrf2 nuclear translocation and regulation of ferroptosis-related proteins. Moreover, inhibition of Nrf2 using ML385 (an Nrf2-specific inhibitor) reversed PTX's neuroprotective effect on MCAO-induced ferroptosis via the SLC7A11/GPX4 signalling pathway. CONCLUSIONS: Ferroptosis is evident following cerebral ischaemia-reperfusion in rats. Pentoxifylline confers protection against IRI in rats by inhibiting ferroptosis through the Nrf2/SLC7A11/GPX4 signalling pathway.

Butylphthalide protects against ischemia-reperfusion injury in rats via reducing neuron ferroptosis and oxidative stress.

Local ischemia in the cerebra leads to vascular injury and necrosis. Ferroptosis is involved in the pathophysiological process of many diseases and widely exists when ischemia-reperfusion injury occurs in many organs. The aim of this study was to evaluate the effect of Butylphthalide (NBP) on middle cerebral artery occlusion (MCAO) rats model-caused neuron injury. Sprague Dawley Rats were randomly allocated to receive sham and MCAO operation. NBP low-dose (40 mg/kg b.w), and high-dose (80 mg/kg b.w) were administrated in MACO rats. Results showed NBP improves infarct volume, attenuates neuronal apoptosis in the brain tissue of MCAO rats. The tumor necrosis factor (TNF-α), IL-6, and malondialdehyde (MDA) levels decreased after NBP administration, while the activity of superoxide dismutase (SOD) and the ratio of GSH/GSSG in MACO rats increased. MACO caused non-heme iron accumulation in the brain tissue and Perl's staining confirmed NBP attenuates ferroptosis in MACO rats. The protein expressions of SCL7A11 and glutathione peroxidase 4 (GPX4) decreased following MCAO, and NBP treatment subsequently increased the expression of SCL7A11 and GPX4. In vitro analysis in cortical neuron cells indicated that the GPX4 inhibitor reverses the inhibition of ferroptosis by NBP, which suggested that the SCL7A11/GPX4 pathway majorly contributed to the NBP ferroptosis protection effect.

Facile construction of a 3D tumor model with multiple biomimetic characteristics using a micropatterned chip for large-scale chemotherapy investigation.

The establishment and application of biomimetic preclinical tumor models for generalizable and high-throughput antitumor screening play a promising role in drug discovery and cancer therapeutics. Herein, a facile and robust microengineering-assisted methodology for highly biomimetic three-dimensional (3D) tumor construction for dynamic and large-scale antitumor investigation is developed using micropatterned array chips. The high fidelity, simplicity, and stability of chip fabrication are guaranteed by improved polydimethylsiloxane (PDMS) microcontact printing. The employment of a PDMS-micropatterned chip permits microscale, simple, biocompatible, and reproducible cell localization with quantity uniformity and 3D tumor array formation with geometric homogeneity. Array-like 3D tumor models possessing complex multilayer cell arrangements, diverse phenotypic gradients, and biochemical gradients were prepared based on the use of easy-to-operate chips. The applicability of the established biomimetic models in temporal and massive investigations of tumor responses to antitumor chemotherapy is also verified experimentally. The results support the importance of the dimensional geometry and biomimetic degree of 3D tumors when conducting antitumor screening to explore drug susceptibility and resistance. This work provides a facile and reliable strategy to perform highly biomimetic tumor manipulation and analysis, which holds great potential for applications in oncology, pharmacology, precision medicine, and tissue microengineering.

Straightforward Cell Patterning with Ultra-Low Background Using Polydimethylsiloxane Through-Hole Membranes.

Micropatterning is becoming an increasingly popular tool to realize microscale cell positioning and decipher cell activities and functions under specific microenvironments. However, a facile methodology for building a highly precise cell pattern still remains challenging. In this study, A simple and straightforward method for stable and efficient cell patterning with ultra-low background using polydimethylsiloxane through-hole membranes is developed. The patterning process is conveniently on the basis of membrane peeling and routine pipetting. Cell patterning in high quality involving over 97% patterning coincidence and zero residue on the background is achieved. The high repeatability and stability of the established method for multiple types of cell arrangements with different spatial profiles is demonstrated. The customizable cell patterning with ultra-low background and high diversity is confirmed to be quite feasible and reliable. Furthermore, the applicability of the patterning method for investigating the fundamental cell activities is also verified experimentally. The authors believe this microengineering advancement has valuable applications in many microscale cell manipulation-associated research fields including cell biology, cell engineering, cell imaging, and cell sensing.

Parallel and large-scale antitumor investigation using stable chemical gradient and heterotypic three-dimensional tumor coculture in a multi-layered microfluidic device.

BACKGROUND: Cancer has been responsible for a large number of human deaths in the 21st century. Establishing a controllable, biomimetic, and large-scale analytical platform to investigate the tumor-associated pathophysiological and preclinical events, such as oncogenesis and chemotherapy, is necessary. METHODS AND RESULTS: This study presents antitumor investigation in a parallel, large-scale, and tissue-mimicking manner based on well-constructed chemical gradients and heterotypic three-dimensional (3D) tumor cocultures using a multifunction-integrated device. The integrated microfluidic device was engineered to produce a controllable and steady chemical gradient by manipulative optimization. Array-like and size-homogeneous production of heterotypic 3D tumor cocultures with in vivo-like features, including similar tumor-stromal composition and functional phenotypic gradients of metabolic activity and viability, was successfully established. Furthermore, temporal, parallel, and high-throughput analyses of tumor behaviors in different antitumor stimulations were performed in a device based on the integrated operations involving gradient generation and coculture. CONCLUSION: This achievement holds great potential for applications in the establishment of multifunctional tumor platforms to perform tissue-biomimetic neoplastic research and therapy assessment in the fields of oncology, bioengineering, and drug discovery.

Nanowired dual-electrodes surface to monitor cerebral ischemia by current-volt measurements.

The level of clotting protein 'factor IX' (FIX) is highly associated with cerebral ischemia, and this research work has developed a sensitive detection of FIX on dielectrode sensor by current-volt measurement. Sensing area was grown with zinc oxide nanowire to attach more probe for FIX interaction. Aptamer was utilized as the detection probe and attached on the sensing electrode surface through amine-aldehyde chemical linkage. In addition, biotin-streptavidin interaction was utilized to attach the higher number aptamers on the electrode surface connected with dual-probe station. FIX detection limit was found as 10 fM in the phosphate buffer saline spiked samples and 1:320 dilution of human serum. The linear ranges were as 10 fM to 100 pM and 1:320 to 1:80, respectively. With a good determination co-efficient [y = 2.6813x - 3.8467; R 2 = 0.9479] this biosensing strategy helps to quantify FIX and monitor the condition of cerebral ischemia.

Advances in Micro/Nanoporous Membranes for Biomedical Engineering.

Porous membrane materials at the micro/nanoscale have exhibited practical and potential value for extensive biological and medical applications associated with filtration and isolation, cell separation and sorting, micro-arrangement, in-vitro tissue reconstruction, high-throughput manipulation and analysis, and real-time sensing. Herein, an overview of technological development of micro/nanoporous membranes (M/N-PMs) is provided. Various membrane types and the progress documented in membrane fabrication techniques, including the electrochemical-etching, laser-based technology, microcontact printing, electron beam lithography, imprinting, capillary force lithography, spin coating, and microfluidic molding are described. Their key features, achievements, and limitations associated with micro/nanoporous membrane (M/N-PM) preparation are discussed. The recently popularized applications of M/N-PMs in biomedical engineering involving the separation of cells and biomolecules, bioparticle operations, biomimicking, micropatterning, bioassay, and biosensing are explored too. Finally, the challenges that need to be overcome for M/N-PM fabrication and future applications are highlighted.

Comprehensive Evaluation of Stable Neuronal Cell Adhesion and Culture on One-Step Modified Polydimethylsiloxane Using Functionalized Pluronic.

Polydimethylsiloxane (PDMS) is a popular and property-advantageous material for developing biomedical microsystems and advancing cell microengineering. The requirement of constructing a robust cell-adhesive PDMS interface drives the exploration of simple, straightforward, and applicable surface modification methods. Here, a comprehensive evaluation of highly stable neuronal cell adhesion and culture on the PDMS surface modified in one step using functionalized Pluronic is presented. According to multiple comparative tests, this modification is sufficiently verified to enable more significant cell adhesion and spreading in both quantity and stability, higher neuronal differentiation and viability/growth, more complete formation of the neuronal network, and stabler neuronal cell culture than the common coating tools on the PDMS substrate. The comparable and even superior cellular effects of this modification on PDMS to the standard coating of polystyrene for in vitro neurological research are demonstrated. Long-term microfluidic neuron culture with stable adhesion and high differentiation on the modified PDMS interface is accomplished, too. The achievement provides a detailed experimental demonstration of this simple and effective modification for strengthening neuronal cell culture on the PDMS substrate, which is useful for potential applications in the fields of neurobiology, neuron microengineering, and brain-on-a-chip.

Enhancement and control of neuron adhesion on polydimethylsiloxane for cell microengineering using a functionalized triblock polymer.

Polydimethylsiloxane (PDMS)-based neuron microengineering provides new opportunities for spatiotemporal control of neuronal activity and stimuli. The demand for long-lasting adhesive PDMS surfaces has steered the development of straightforward, feasible, and accessible interface modifications. Here, we describe an innovative approach for promoting and engineering neuron adhesion on a PDMS substrate based on a very simple modification using poly-d-lysine-conjugated Pluronic F127, a functionalized triblock polymer. The modification procedure only involves single-step pipetting or microfluidic-guided introduction for the reinforcement of cell adhesion in quantity, extensibility, and stability. Micropatterning at a single-cell resolution, microfluidic long-term culture, and neuron network formation were achieved. The present approach provides a previously unprecedented simple and effective technique for neuron adhesion on PDMS and may be useful for applications in neurobiology, tissue engineering, and neuronal microsystems.

Antitumor activity of cobrotoxin in human lung adenocarcinoma A549 cells and following transplantation in nude mice.

The aim of the present study was to investigate cobra neurotoxin (cobrotoxin) activity in A549 cell lines transplanted into nude mice, and to explore its molecular mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the growth inhibition rate of cobrotoxin in human lung A549 adenocarcinoma cells and HFL1 lung fibroblasts. Cell colony formation assays were performed to determine the effect of cobrotoxin on A549 cell colony formation, and transmission electron microscopy was used to detect cobrotoxin autophagy. In addition, western blot analysis was performed to determine the effect of 3-methyl adenine (3-MA) activity on the inhibition of autophagy, SB203580 inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway, and Beclin 1, LC3, p62, p38 and phosphorylated (p)-p38 protein expression. Nude mice were injected with human lung A549 cells, and intervention and control groups were compared with regard to tumor suppression. The MTT assay revealed that various concentrations of cobrotoxin inhibited growth of A549 cells, but not HFL1 cells. A549 cell colony formation decreased and autophagosome activity was significantly increased compared with the controls. Following 3-MA administration, SB203580 autophagosome activity decreased, and following cobrotoxin administration, Beclin 1, p-p38, and LC3-II protein expression significantly increased, whereas p62 expression significantly decreased. Following 3-MA inhibition of autophagy, Beclin 1, LC3-II and p62 expression increased. Furthermore, following SB203580 inhibition of the p38-MAPK pathway, Beclin 1, p-p38, LC3-II and p62 protein expression increased. Cobrotoxin exhibited inhibitory activity on the human lung cancer A549 cells transplanted into the nude mice, suppressing the tumor growth rate by 43.4% (cobrotoxin 40 µg/kg group). However, following the addition of 3-MA (10 mmol/kg) and SB203580 (5 mg/kg), the suppression of the tumor growth rate decreased significantly. Cobrotoxin inhibits the growth of human lung cancer A549 cells in vitro and A549 cells transplanted into nude mice. Furthermore, the induction of autophagy may be associated with the activation of the p38-MAPK pathway.