The Antitumor Mechinsms Of Constructed And Gate Genetic Circuit Based On Heat Shock Response Transduction

Objective: The major obstacle of clinical gene therapy is lacking ofoptimal techniques for transferring genes to targeted tumor tissues. Currentvectors such as viral or non-viral vectors can limit gene transfer to tumortissues, but surrounding tissues often receive unwanted genes andoverexpression of therapeutic genes there can induce damage to normaltissues. Therefore, targeted gene therapy technologies that can selectivelyregulate therapeutic gene expression within the tumor tissues and producemarked antitumor effects require enrichment steps.In our study, through combination the characteristics of radiationsensitivity with hypoxia in solid tumors, we proposed to devise a specialvector which could be activated by radiation and hypoxia together but noteither alone. This strategy could regulate therapeutic gene expression in hypoxic tumor cells in radiation field instead of peritumoral and otherhypoxic normal tissues and accomplished targeted treatment. And throughtargeted regulation of ectogenic gene, this designed gene therapy couldeffectively avoid normal tissues toxicity and reached ideal antitumor effects.The purpose of this research was to construct an AND gate genetic circuitthrough connecting the elements in the heat shock response pathway withradiation sensitivity promoter cArG.Methods: Through application of DNA synthesizer, cArG6promoterand HSE4reporter were synthesized by complementaryoligodeoxyribonucleotides reannealing technology which could retainnative activity of the constructed sequences and subcloned intotranscriptional control subsequences in5'-LTR sequence of plxsn. SNF1,HSF1and wtp53cDNA sequences were generated by proofreading PCRfrom puc18, pOTB7and pCMV6-XL4plasmids respectively and verifiedby sequencing. SNF1and HSF1genes were inserted into downstream ofcArG6and control vectors named plxsn-EGFP were formed. Therapeuticvectors named plxsn-wtp53were constructed by replacing EGFP withwtp53gene. All fragments of recombinant vectors were comparedsequences by BLAST and identified by1%agarose gel electrophoresisstained with Gold ViewTMNuclear Acid.Results: The DNA sequences of the two vectors were confirmedidentically to purpose genes by BLAST. Addtionally, through enzyme incision technology, fragments of recombinated vectors were analyzed. Thebands (7800bp,9600bp and11000bp) that were cut off through usingEcoRI, AgeI and BamHI restriction enzymes respectively were consistentwith the lengths of plxsn-SNF1, plxsn-EGFP and plxsn-wtp53genes. Andthe bands of double enzymatic solution (2300bp,3000bp and1200bp)through using NotI and EcoRI, EcoRI and AgeI, and BamHI and EcoRIwere also consistent with the lengths of SNF1, HSF1and wtp53genes.Conclusions:1. Genetic mutation cannot be found in the constructed AND gategenetic circuit through sequencing technology.2. The construction of the AND gate vectors is successful throughrestriction enzyme measurements. Objective: In our experiments, we further identified if the constructedAND gate could be activated under radiation and hypoxia dual stimuliinstead of either alone.Methods: A549cells were transciently transfected byplxsn-EGFP/wtp53vectors and then received radiation or hypoxia treatment. The EGFP expression was observed through fluorescencemicroscope and levels of SNF1,HSF1and p53proteins were checked outthrough western-blotting technology.Results: Dynamic alterations of EGFP expression were observed inA549cells. As expected, EGFP only expressed in transfected A549cellsreceiving radiation and hypoxia stimuli while apparent EGFP protein wasnot detected in other treatment cells. And EGFP level changed in atime-dependent manner. The lowest level of EGFP protein exhibited at24hand the highest level was observed at72h. The activation process of theAND gate was further confirmed through SNF1, HSF1and p53proteinsexpression in transfected A549cells. Maximal SNF1proteins weredetectable in cells treated with radiation or radiation and hypoxia aftertransfection of plxsn-wtp53. And apparent SNF1protein expression wasnot examined in normal groups regardless of receiving treatments or not forits yeast source. Similar result of HSF1protein induction was found intransfected A549cells. Unexpectedly, although maximal levels of p53protein expressed in transfected cells receiving radiation and hypoxiastimuli, minor elevated p53protein also appeared in cells receivingradiation treatment which should maintained a low level as other groups.Conclusions:1. Our synthetic AND gate vectors could be activated only underradiation and hypoxia instead of either alone. 2. The transgene expression was indeed due to activation of radiationresponse promoter cArG6under radiation stimulus through western-blottinganalysis.3. The western-blotting result identified that radiation could inducep53expression which was consistent with previous researches. Objective: In our experiments, we further evaluated the antitumorefficacy of the constructed AND gate to provide a novel and effectivestrategy for clinical solid tumor treatments.Methods: A549cells were transiently transfected by plxsn-wtp53vectors and then collected at24h,48h and72h. Apoptosis and cell cycledetection were measured by Flow Cytometry and proliferation of cells wastested by CCK-8. A549cells were harvested from subconfluent cell cultureplates and resuspended in PBS at the concentration of5×107cells permilliliter, then injected with200μl/spot into either flank side of the micesubcutaneously. The mice were monitored by measuring tumor growth andbody weight every two days. Tumor volumes were calculated from 2-dimensional measurements by the formula: tumor volume=length×width~2/2. When bearing tumors volume reached60mm3and bodyweight reached18grams, mice were treated by injection ofplxsn-EGFP/wtp53plasmids (20μg/kg) intratumorally and focal2Gy ofradiation at the following day for3times. Then animals were killed at Day3after treatment. Xenografts were dissected free of surrounding normaltissues and fixed with4%paraformaldehyde. Then the experiments wereproceeded as following: H＆E staining, immunohistochemisty assays forwtp53expression and apoptotic cells in A549xenografts.Results:1. Compared to other groups, treatment group containing activatedp53caused G1-G2phase cell cycle arrest.2. In A549cells transfected plxsn-wtp53and receiving radiation andhypoxia, activated wtp53induced significant apoptosis effects at48and72h and the apoptosis rate was higher than other control groups (P<0.01).3. A549cells were interfered with plxsn-wtp53vectors, and thenreceived radiation or hypoxia treatments. As expected, significant tumorproliferation inhibition was observed in wp53+radiation+hypoxia group.The proliferation in this group was decreased significantly compared toother groups after treatments at48and72h (P<0.05).4. Significant tumor regression was observed in the tumors receivingplxsn-wp53injection and radiation. Although xenografts grew slightly during the6days of treatments, from60mm3to70mm3, the mean volumekept decreasing along with time and the volume became59mm3on Day13and the volume is significantly smaller than other control groups (P<0.05).5. The H＆E staining analysis showed that cells in A549xenograftswhich received plxsn-wtp53injection and radiation treatments representedsignificant apoptosis behaviors, such as cell shrinks, karyopyknosis,bubbles and obvious apoptotic bodies in cytoplasm.6. Moreover, immunohistochemical detection showed that p53proteinlocated in nuclear envelops and perinuclear space and exhibited higherexpression level in xenografts which received plxsn-wtp53injection andradiation treatments (P<0.01) than other groups although minor wtp53protein could be detected in control groups.7. Through TUNEL assays, prominent apoptotic response inxenografts which received plxsn-wtp53injection and radiation treatmentswas detected and the number of positive cells was much higher than thatreceiving other treatments (P<0.01) while no obvious apoptosis andnecrosis cells were observed in the normal tumor mass.8. The HIF-1α protein expression was detected in A549xenografts tovalidate that the existence of hypoxic areas. HIF-1α protein, an importanthypoxic marker, could be detected in all A549xenografts regardless ofreceiving treatments or not and there was no discernible difference in theamount and specificity in different groups. Conclusions:1. Activated AND gate produced a marked effect on mitosis arrest,apoptosis promotion and proliferation progression inhibition to A549cells.2. Although plasmids injection or radiation could induce minorapoptosis, AND gate could be activated in vivo by6Gy of radiation andtake effective antitumor effects which were consistent with the experimentsin vitro.3. The HIF-1α expression demonstrated that hypoxic districts existedin A549xenografts. Objective: Different tumors' responses to radiation vary, and eventumors from the same histological group can also be present with extremelyheterogeneous radiosensitivity. Moreover, the growth of most malignanttumors shows irregular patterns, and some tumors are closely linked withvital organs such as brain, kidney or nerve, clinical treatment dose ofradiation should be attenuated to reduce the damage to normal tissues. So,the effect of the AND gate constructed previously may be weakened bydifferent degrees. In present research, we proposed to explore if the AND gate can be activated by low-dose radiation and regulated by radiationdoses and further demonstrated the feasibility of the clinical use of ourAND gate.Methods: The apoptosis proportion of293and A549cells receivingdifferent radiation-dose treatments was detected through Flow Cytometryand the apoptosis rate of the two cell lines induced by2Gy and6Gy ofradiation was analyzed. The EGFP expression in A549, HeLa, HepG2andMCF-7cells receiving different treatments was observed. A549cells weretransfected by plxsn-wtp53vectors and cellular morphology was observed.p53mRNA and protein were detected through RT-PCR andwestern-blotting technologies. Cell cycle and apoptosis were tested byFlow Cytometry and proliferation of A549cells was tested by CCK-8assay.Apoptosis related factors BAX and BCL-2proteins were identified throughwestern-blotting. A549cells were harvested from subconfluent cell cultureplates and resuspended in PBS at the concentration of5×107cells permilliliter, then injected with200μl/spot into either flank side of the micesubcutaneously. The mice were monitored by measuring tumor growth andbody weight every two days. When bearing tumors volume reached60mm3and body weight reached18grams, mice were treated by injection ofplxsn-EGFP/wtp53plasmids (20μg/kg) intratumorally and focal radiationof2Gy at the following day. Animals were killed at Day3after treatment.Xenografts were dissected free of surrounding normal tissues and fixed with4%paraformaldehyde. Then the experiments were proceeded asfollowing: H＆E staining for detection of apoptotic cells,immunohistochemisty assays for EGFP and wtp53expression andapoptotic cells in A549xenografts, immunofluorescence dual staining forHIF-1α and p53expression.Results:1.293and A549cells showed a lower apoptosis level under2Gy ofradiation compared with other radiation doses. Therefore, we chose2Gy asthe stimulus dose, and tested whether it could initiate our constructed ANDgate gene circuit. Additionally, we could also see that tumor cells weremore resistant to radiation and hypoxia increased the ability while oppositerole was showed in293cells.2. A549cells were transiently transfected with plxsn-EGFP vectorsand the activity of the vectors was assessed by EGFP expression. We couldsee that EGFP only expressed in a low level in transfected cells receivingradiation and hypoxia instead of others at24h, but its expression levelapparently elevated at48h and persisted at72h.3. In wtp53group, p53mRNA expression which was detected byRT-PCR displayed a lower level at24h but much higher at48h (P<0.01)and72h (P<0.01) compared with that in the normal and control groups.4. Similar expression of wtp53protein was detected bywestern-blotting. wtp53protein showed much higher levels at48h (P<0.01) and72h (P<0.01) in the wtp53group, compared with no obviousexpression in the normal and control groups. Reason of no wtp53band inwtp53group at24h might be low expression level of p53that could not bedetected.5. From the bright fields of A549cells, we could observe plxsn-wtp53vectors transfected cells receiving2Gy of radiation and hypoxia treatmentsshowed significant growth inhibition and apoptosis promotion, such asobvious intercellular space and numbers of cells decreasing. However, cellsin the normal and control groups displayed no obvious morphologicalchanges.6. A549cells were interfered in the plxsn-wtp53vectors, and thenreceived radiation or hypoxia treatments. As expected, significant tumorproliferation inhibition was observed in wp53group. The proliferation inthis group was decreased significantly compared to other groups aftertreatments at24and48h (P<0.05). No significant difference of the threegroups at72h was because of the aggressive proliferation of A549cells.7. Compared to other groups, treatment group containing activatedp53caused G1-G2phase cell cycle arrest at48h.8. In A549cells transfected plxsn-wtp53and receiving radiation andhypoxia, activated wtp53induced significant apoptosis effects at24,48and72h and the apoptosis rate was higher than other treatment groups (P<0.01).9. After48h, level of proapoptosis protein BAX and prosurvival protein BCL-2in A549cells transfected with plxsn-wtp53vectors orreceiving radiation and hypoxia treatments was tested throughwestern-blotting technology. And higher BAX and lower BCL-2wereobserved in wtp53group compared to other groups (P<0.01).10. The distribution of plxsn-EGFP plasmids in A549xenografts wasmonitored. Remarkably, we found the activated AND gate marker EGFPonly preferentially expressed in xenografts of plxsn-EGFP injection andreceiving2Gy of radiation while no EGFP expression was observed inother groups.11. The H＆E staining analysis showed that cells in A549xenograftswhich received plxsn-wtp53injection and radiation treatments representedsignificant apoptosis behaviors, such as cell shrinks, karyopyknosis,bubbles and obvious apoptotic bodies in cytoplasm. And minor apoptosiscells were observed in xenografts receiving radiation treatment.12. Through TUNEL assays, prominent apoptotic response inxenografts which received plxsn-wtp53injection and radiation treatmentswas detected and the number of positive cells was much higher than thatreceiving other treatments (P<0.01) and no obvious apoptosis and necrosiscells were observed in the normal tumor mass.13. Moreover, immunohistochemical detection showed that p53protein located in nuclear envelops and perinuclear space and exhibitedhigher expression level in xenografts which received plxsn-wtp53injection and radiation treatments (P<0.01) than other groups although minor wtp53protein could be detected in other control groups.14. We checked the expression of HIF-1α/wtp53throughimmunofluorescence double staining. HIF-1α protein, an importanthypoxic marker, could be detected in all A549xenografts regardless ofreceiving radiation treatment or not and there was no discernible differencein the amount and specificity among different groups. But the wtp53protein showed different expression. wtp53protein was detected tospecifically express in HIF-1α positive cells of xenografts receiving2Gy ofradiation instead of other groups.15. To further test whether the AND gate could be activated in othertumor cells or not, HeLa, HepG2and MCF-7cells were transientlytransfected with plxsn-EGFP vectors and then treated with2Gy of radiationor1%O2or both of them. EGFP protein was observed to only express incells receiving radiation and hypoxia instead of cells receiving othertreatments at48h.Conclusions:1. AND gate genetic circuit can be activated by2Gy and1%O2.2. The constructed AND gate can be regulated by radiation dose.3. Activated AND gate induces apparent antitumor effects and minornormal tissue damage compared to previous research.