In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter

Introduction

Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using ¹²⁵I and ¹³¹I therapy in vivo.

Methods

We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; ¹²⁵I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with ¹³¹I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.

Results

qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more ¹²⁵I than CNE-2Z cells and xenografts. In vitro, ¹³¹I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, ¹³¹I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of ¹³¹I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.

Conclusion

Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.     

Synthesis and bioevaluation of radioiodinated nitroimidazole hypoxia imaging agents by one-pot click reaction

Eight radioiodinated 2-nitroimidazole derivatives for use as hypoxia imaging agents were synthesized by one-pot click reaction using four azides, two alkynes, and [¹³¹I]iodide ions and evaluated by hypoxic cellular uptake and biodistribution experiments. The results suggested that radiotracers with suitable partition coefficients (log P: −0.2–1.2) were more likely to have higher hypoxic cellular uptake. Among these eight molecules, [¹³¹I]15 ([¹³¹I]-(5-iodo-1-(2-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)ethyl)-4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazole)) had a suitable log P (0.05 ± 0.03) and contained two 2-nitroimidazole groups. The hypoxic/aerobic cellular uptake ratio of [¹³¹I]15 was 4.4 ± 0.5, and the tumor/blood (T/B) and tumor/muscle (T/M) ratios were 2.03 ± 0.45 and 6.82 ± 1.70, respectively. These results suggested that [¹³¹I]15 was a potential hypoxia imaging agent.     

Tissue distribution of 131I radiolabeled transferrin in the athymic nude mouse: Localization of a human colon adenocarcinoma HT-29 xenograft

The tissue distribution of ¹³¹I-transferrin (¹³¹I-Tf) was studied in athymic nude mice having s.c. human colonic adenocarcinoma HT-29 xenografts. Four days after ¹³¹I-Tf injection, the ¹³¹I specific activity measured in the HT-29 tumor, i.e. amount of radioactivity per gram of fresh tissue, represented 0.31 ± 0.09% of the injected radioactivity and was 1.90 fold more than that measured in the murine colon (P < 0.05). After correction for intravascular ¹³¹I-Tf as estimated by means of ^99mTc-Sn in vivo labeling of red blood cells, the ¹³¹I specific activity observed in the HT-29 tumor was 7.21 fold more than that observed in the murine colon. This subtracting method enabled us to localize a HT-29 tumor xenograft by γ scintigraphy of the entire animal and demonstrated that ¹³¹I-Tf could be a non-specific but potent marker for human colon cancer.     

BRMS1 Suppresses Glioma Progression by Regulating Invasion,Migration and Adhesion of Glioma Cells

Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene in several solid tumors. However, the expression and function of BRMS1 in glioma have not been reported. In this study, we investigated whether BRMS1 play a role in glioma pathogenesis. Using the tissue microarray technology, we found that BRMS1 expression is significantly decreased in glioma compared with tumor adjacent normal brain tissue (P<0.01, χ² test) and reduced BRMS1 staining is associated with WHO stages (P<0.05, χ² test). We also found that BRMS1 was significantly downregulated in glioma cell lines compared to normal human astrocytes (P<0.01, χ² test). Furthermore, we demonstrated that BRMS1 overexpression inhibited glioma cell invasion by suppressing uPA, NF-κB, MMP-2 expression and MMP-2 enzyme activity. Moreover, our data showed that overexpression of BRMS1 inhibited glioma cell migration and adhesion capacity compared with the control group through the Src-FAK pathway. Taken together, this study suggested that BRMS1 has a role in glioma development and progression by regulating invasion, migration and adhesion activities of cancer cells.     

HORMAD2 methylation‐mediated epigenetic regulation of gene expression in thyroid cancer

This study is aimed to investigate the methylation level of candidate genes and its impact on thyroid carcinoma (THCA) development. Infinium Human Methylation 450 BeadChip Arrays by Illumina (Illumina HM450K) was the most popular CpG microarray platform widely used in biological and medical research. The methylation level of differentially expressed genes and their corresponding CpG sites were analysed by R programme. The expression of HORMAD2 was evaluated by qRT‐PCR and Western blot, while the methylation level was examined via methylation‐specific PCR. Cell viability, metastasis, cell cycle and apoptosis were detected by MTT assay, transwell and wound healing assay and flow cytometry, respectively, after treatment with 5‐aza‐2′‐deoxycytidine (5‐Aza). Tumour formation assay was used to analyse thyroid tumour growth in nude mice in vivo. The methylation levels of all 116 differentially expressed genes were analysed. HORMAD2 was significantly hypermethylated and its mRNA expression was inhibited in THCA cells. After treatment with 5‐Aza, HORMAD2 expression was up‐regulated in THCA cells and its overexpression can suppress thyroid cancer cell viability, mobility and invasiveness remarkably. Up‐regulation of HORMAD2 in THCA cells could prolong G0/G1 phase and shorten S phase to impede cell mitosis as well as promote thyroid cancer cells apoptosis. Furthermore, tumour formation assay showed that increased HORMAD2 level impeded tumour growth in vivo. Hypermethylation of HORMAD2 could induce THCA progression, while hypomethylation of HORMAD2 retard cell growth and mobility and facilitate apoptosis through increasing its mRNA expression.     

FYN promotes gastric cancer metastasis by activating STAT3-mediated epithelial-mesenchymal transition

Gastric cancer is one of the most lethal cancers worldwide. FYN, a gene that is differentially expressed in gastric cancer, is considered a critical metastasis regulator in several solid tumors, but its role in gastric cancer is still unclear. This study aimed to evaluate the role of FYN and test whether FYN promotes migration and invasion of gastric cancer cells in vitro and in vivo via STAT3 signaling. FYN was overexpressed in gastric cancer and positively correlated with metastasis. FYN knockdown significantly decreased cancer cell migration and invasion, whereas FYN overexpression increased cancer migration and invasion. Genetic inhibition of FYN decreased the number of metastatic lung nodules in vivo. Several epithelial-mesenchymal transition markers were positively correlated with FYN expression, indicative of FYN involvement in this transition. Furthermore, gene set enrichment analysis of a Cancer Genome Atlas dataset revealed that the STAT3 signaling pathway was positively correlated with FYN expression. STAT3 inhibition reversed the FYN-mediated epithelial-mesenchymal transition and suppressed metastasis. In conclusion, FYN promotes gastric cancer metastasis possibly by activating STAT3-mediated epithelial mesenchymal transition and may be a novel therapeutic target for gastric cancer.     

Downregulation of m6A Reader YTHDC2 Promotes the Proliferation and Migration of Malignant Lung Cells via CYLD/NF-κB Pathway

Lung cancer is one of the most common types of carcinoma worldwide. Cigarette smoking is considered the leading cause of lung cancer. Aberrant expression of several YT521-B homology (YTH) family proteins has been reported to be closely associated with multiple cancer types. The present study aims to evaluate the function and regulatory mechanisms of the N6-methyladenosine (m⁶A) reader protein YTH domain containing 2 (YTHDC2) by in vitro, in vivo and bioinformatics analyses. The results revealed that YTHDC2 was reduced in lung cancer and cigarette smoke-exposed cells. Notably, bioinformatics and tissue arrays analysis demonstrated that decreased YTHDC2 was highly associated with smoking history, pathological stage, invasion depth, lymph node metastasis and poor outcomes. The in vivo and in vitro studies revealed that YTHDC2 overexpression inhibited the proliferation and migration of lung cancer cells as well as tumor growth in nude mice. Furthermore, YTHDC2 decreased expression was modulated by copy number deletion in lung cancer. Importantly, the cylindromatosis (CYLD)/NF-κB pathways were confirmed as the downstream signaling of YTHDC2, and this axis was mediated by m⁶A modification. The present results indicated that smoking-related downregulation of YTHDC2 was associated with enhanced proliferation and migration in lung cancer cells, and appeared to be regulated by DNA copy number variation. Importantly, YTHDC2 functions as a tumor suppressor through the CYLD/NF-κB signaling pathway, which is mediated by m⁶A modification.     

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth,Angiogenesis, and Metastasis

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1^−/−) mice, we show that prostaglandin E₂ (PGE₂) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE₂ production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE₂ further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE₂ production by the osteoblasts. PGE₂, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE₂ acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4^−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE₂ effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE₂ signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.     

Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression

Overexpression of histone deacetylases (HDACs) in cancer commonly causes resistance to genotoxic-based therapies. Here, we report on the novel mechanism whereby overexpressed class I HDACs increase the resistance of glioblastoma cells to the S_N1 methylating agent temozolomide (TMZ). The chemotherapeutic TMZ triggers the activation of the DNA damage response (DDR) in resistant glioma cells, leading to DNA lesion bypass and cellular survival. Mass spectrometry analysis revealed that the catalytic activity of class I HDACs stimulates the expression of the E3 ubiquitin ligase RAD18. Furthermore, the data showed that RAD18 is part of the O⁶-methylguanine-induced DDR as TMZ induces the formation of RAD18 foci at sites of DNA damage. Downregulation of RAD18 by HDAC inhibition prevented glioma cells from activating the DDR upon TMZ exposure. Lastly, RAD18 or O⁶-methylguanine-DNA methyltransferase (MGMT) overexpression abolished the sensitization effect of HDAC inhibition on TMZ-exposed glioma cells. Our study describes a mechanism whereby class I HDAC overexpression in glioma cells causes resistance to TMZ treatment. HDACs accomplish this by promoting the bypass of O⁶-methylguanine DNA lesions via enhancing RAD18 expression. It also provides a treatment option with HDAC inhibition to undermine this mechanism.Subject terms: Acetylation, Oncogenes     

The detection of virally induced tumors by 131I- and 125I-labeled syngeneic monoclonal antibodies

Summary The binding of the syngeneic monoclonal antibodies IC5F5 and 4D2B4 to Rauscher virus-induced myeloid leukemic (RMB-1) cells was analyzed in vivo in tumor-bearing BALB/c mice. To verify it these antibodies bind specifically to RMB-1 cells, purified antibodies were iodinated with the isotopes ¹²⁵I and ¹³¹I. Mice previously inoculated with tumor cells were injected with these labeled monoclonal antibodies and the plasma clearance and the tissue distribution were determined. The clearance in tumor-bearing animals was faster than in control mice. The tissue distribution was corrected for nonspecific accumulation by scoring for an unrelated antibody. Calculation of a localization index showed that IC5F5 binds at least 4.5 times more specifically to tumor cells than to other tissues. A preferential localization of radioactivity in s.c. tumor tissue was seen in the scanning of animals injected with ¹³¹I-labeled antibodies. The most direct proof of specific binding was observed in autoradiograms of animals treated with ¹²⁵I-labeled antibodies. Small islands of tumor cells in the livers of mice inoculated i.v. had a high density of grains compared to other tissues and also compared to tumor cells in mice treated with unrelated monoclonal antibodies. These results show efficient targeting of these monoclonal antibodies and make immunotherapy of these myeloid leukemic cells possible.     

Small proline-rich protein-1B is overexpressed in human oral squamous cell cancer stem-like cells and is related to their growth through activation of MAP kinase signal

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1^br) cells by the ALDEFLUOR assay. Small proline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1^br cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1^br cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4.     

The calcium pump PMCA4 prevents epithelial-mesenchymal transition by inhibiting NFATc1-ZEB1 pathway in gastric cancer

Epithelial-mesenchymal transition (EMT) is considered as the key mechanism involved in cancer metastasis. Several studies showed that various cell membrane calcium channels play different roles in cancer metastasis. In the present study, the potential role of ATPase plasma membrane Ca²⁺ transporting 4 (PMCA4) in regulating EMT in gastric cancer (GC) was investigated. GC patients who underwent radical surgery were enrolled in this study. In vitro human GC cell lines MKN45 and NCI-N87 were used, and MKN45 cells were injected in nude mice to evaluate tumor development. Our results showed that low PMCA4 expression was associated with advanced TNM stage and poor prognosis in GC patients. Knockdown of PMCA4 suppressed E-cadherin, grainyhead like 2 (GRHL2) and ovo-like 1 (OVOL1) expression, up-regulated vimentin expression, increased migration and invasion ability, and promoted the resistance to cytotoxic drug. Furthermore, GC cells displayed an elongated fibroblastoid morphology when PMCA4 was knockdown. PMCA4 overexpression resulted in an up-regulated E-cadherin expression and decreased migration and invasion ability. In vivo metastasis assay showed that PMCA4 overexpression resulted in a decreased incidence of lung metastasis. PMCA4 inhibition increased ZEB1 expression and nuclear accumulation of nuclear factor of activated T-cell isoform c1 (NFATc1). EMT induced by PMCA4 inhibition could be prevented by the knockdown of NFATc1 or ZEB1. In addition, cyclosporine A prevented EMT induced by PMCA4 inhibition by suppressing the NFATc1-ZEB1 pathway. Our data identified a novel mechanism in the regulation of EMT in GC, and provided a novel target in the treatment of EMT subtype in GC.     

Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas

The human Matrix MetalloProtease-9 (hMMP-9) is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B), fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG) and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of ^99mTc-MAG-aptamer and ¹¹¹In-DOTA-F3B. ^99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of ¹¹¹In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05) higher for ¹¹¹In-DOTA-F3B with 2.0%ID/g than for the ¹¹¹In-DOTA-control oligonucleotide (0.7%ID/g) with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.     

Cannabinoids act as necrosis-inducing factors in Cannabis sativa

Cannabis sativa is well known to produce unique secondary metabolites called cannabinoids. We recently discovered that Cannabis leaves induce cell death by secreting tetrahydrocannabinolic acid (THCA) into leaf tissues. Examinations using isolated Cannabis mitochondria demonstrated that THCA causes mitochondrial permeability transition (MPT) though opening of MPT pores, resulting in mitochondrial dysfunction (the important feature of necrosis). Although Ca²⁺ is known to cause opening of animal MPT pores, THCA directly opened Cannabis MPT pores in the absence of Ca²⁺. Based on these results, we conclude that THCA has the ability to induce necrosis though MPT in Cannabis leaves, independently of Ca²⁺. We confirmed that other cannabinoids (cannabidiolic acid and cannabigerolic acid) also have MPT-inducing activity similar to that of THCA. Moreover, mitochondria of plants which do not produce cannabinoids were shown to induce MPT by THCA treatment, thus suggesting that many higher plants may have systems to cause THCA-dependent necrosis.Key words: cannabinoid, Cannabis sativa, cylophilin D, mitochondrial permeability transition, necrosisCannabis sativa produces unique secondary metabolites consisting of alkylresorcinol and monoterpene groups.¹ These metabolites called cannabinoids are well known to show a variety of interesting pharmacological activities including psychoactive effect and analgesic effect. Therefore, cannabinoids have attracted a great deal of attention, whereas why C. sativa produces such metabolites has long remained unclear. However, we have recently obtained evidences indicating the physiological function of THCA in Cannabis leaves.²We discovered that THCA is stored in capitate-sessile glands on Cannabis leaves and that secretion of this cannabinoid into leaf tissues causes cell death. When the properties of THCA were examined using cultured Cannabis cells, this cannabinoid induced plasmamembrane shrinkage and DNA degradation. These responses are regarded as the features of apoptotic cells, but were not suppressed by apoptosis inhibitors. In contrast, the necrosis inhibitor cyclosporine A significantly inhibited both plasmamembrane shrinkage and DNA degradation in Cannabis cells. Therefore, we assumed that THCA induces necrotic cell death in Cannabis cells and leaves.Necrosis in plants and animals is usually triggered by MPT though opening of MPT pores.^3,4 MPT is known to cause mitochondrial dysfunction by mitochondrial swelling and loss of mitochondrial membrane potential (ΔΨm),^5,6 and we also confirmed that THCA induces mitochondrial swelling and ΔΨm reduction in mitochondria isolated from Cannabis cells and that pretreatment with cyclosporine A inhibits both responses. Based on these evidences, we concluded that THCA has the activity to induce MPT-dependent necrosis.As described above, MPT pores play an important role in necrosis induction, whereas the mechanism of their opening in higher plants has not been fully understood. However, binding of cyclophilin D (a protein present in mitochondrial matrix) to MPT pores is shown to be essential for their opening in plants as well as animal.^7–9 In animal mitochondria, Ca²⁺ mediates this binding reaction, leading to opening of MPT pores. Wheat mitochondria are also shown to undergo swelling through opening of MPT pores in response to Ca²⁺,⁹ whereas MPT pores of oats,¹⁰ Arabidopsis thaliana¹¹ and C. sativa² do not open by Ca²⁺ treatment. In contrast, THCA catalyzed opening of Cannabis MPT pores in the absence of Ca²⁺, suggesting that THCA directly mediates binding of cyclophilin D to MPT pores (Fig. 1). In addition, we have now confirmed that THCA causes dysfunction though MPT in mitochondria of plants (rice, soybean, A. thaliana and Scutellaria baicalensis) lacking cannabinoid-producing ability (data not shown). Therefore, many higher plants may have the systems to induce THCA-dependent necrosis.Open in a separate windowFigure 1A model depicting the opening mechanism of MPT pores in mitochondria. CYD, cyclophilin D; CN, cannabinoid.Furthermore, we investigated whether other cannabinoids and their related compounds can mediate MPT in Cannabis mitochondria. When the MPT-inducing activity of each sample was measured by monitoring both ΔΨm reduction (Fig. 2) and mitochondrial swelling (data not shown), we confirmed that cannabinoids tested here (cannabidiolic acid and cannabigerolic acid) possess the activities similar to those of THCA. On the other hand, olivetolic acid (the akylresorcinol moiety of cannabinoid) and geraniol (the monoterpene moiety of cannabigerolic acid) showed neither ΔΨm reduction nor mitochondrial swelling (Fig. 2). These results suggested that the structures (cannabinoid skeleton) where monoterpene and olivetolic acid are coupled to each other seem essential for opening of MPT pores. Therefore, we assumed that plant cyclophilin D and MPT pores have the cannabinoid-binding site.Open in a separate windowFigure 2Change of ΔΨm by treatment with various compounds (A) and their chemical structures (B). The isolated mitochondria were stained with the ΔΨm-indicating reagent (tetramethylrhodamine methylester, TMRM) and then incubated with 200 µM of each compound for 60 min. The intensity of TMRM fluorescence was measured using a fluorescence microplate reader. A decrease of the fluorescence intensity indicates ΔΨm reduction. CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; OLA, olivetolic acid.Plant cell death is shown to participate in important physiological responses such as leaf senescence, somatic embryogenesis and defense against microbial pathogens.^12,13 Based on its induction mechanism, plant cell death is largely classified into apoptosis and necrosis. Although the molecular mechanism of apoptosis has been extensively investigated, there is little precise information on plant necrosis. However, our study would provide important insight into necrosis-inducing mechanisms in higher plants.