A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis
IntroductionWe have demonstrated that transposons derived from ctDNA can be transferred between cancer cells. The present research aimed to investigate the cellular uptake and intracellular trafficking of Multiple Myeloma-zip code (MM-ZC), a cell-specific zip code, in myeloma cell lines. We demonstr...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-02-01
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| Series: | Frontiers in Oncology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fonc.2024.1288724/full |
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| author | Pavan Kumar Puvvula Lourdes Martinez-Medina Munevver Cinar Lei Feng Andrey Pisarev Anthony Johnson Leon Bernal-Mizrachi |
| author_facet | Pavan Kumar Puvvula Lourdes Martinez-Medina Munevver Cinar Lei Feng Andrey Pisarev Anthony Johnson Leon Bernal-Mizrachi |
| author_sort | Pavan Kumar Puvvula |
| collection | DOAJ |
| description | IntroductionWe have demonstrated that transposons derived from ctDNA can be transferred between cancer cells. The present research aimed to investigate the cellular uptake and intracellular trafficking of Multiple Myeloma-zip code (MM-ZC), a cell-specific zip code, in myeloma cell lines. We demonstrated that MM-ZC uptake by myeloma cells was concentration-, time- and cell-type-dependent.MethodsFlow cytometry and confocal microscopy methods were used to identify the level of internalization of the zip codes in MM cells. To screen for the mechanism of internalization, we used multiple inhibitors of endocytosis. These experiments were followed by biotin pulldown and confocal microscopy for validation. Single interference RNA (siRNA) targeting some of the proteins involved in endocytosis was used to validate the role of this pathway in ZC cell internalization.ResultsEndocytosis inhibitors identified that Monensin and Chlorpromazine hydrochloride significantly reduced MM-ZC internalization. These findings suggested that Clathrin-mediated endocytosis and endosomal maturation play a crucial role in the cellular uptake of MM-ZC. Biotin pulldown and confocal microscopic studies revealed the involvement of proteins such as Clathrin, Rab5a, Syntaxin-6, and RCAS1 in facilitating the internalization of MM-ZC. Knockdown of Rab5a and Clathrin proteins reduced cellular uptake of MM-ZC and conclusively demonstrated the involvement of Clathrin-Rab5a pathways in MM-ZC endocytosis. Furthermore, both Rab5a and Clathrin reciprocally affected their association with MM-ZC when we depleted their proteins by siRNAs. Additionally, the loss of Rab5a decreased the Syntaxin-6 association with MMZC but not vice versa. Conversely, MM-ZC treatment enhanced the association between Clathrin and Rab5a.ConclusionOverall, the current study provides valuable insights into the cellular uptake and intracellular trafficking of MM-ZC in myeloma cells. Identifying these mechanisms and molecular players involved in MM-ZC uptake contributes to a better understanding of the delivery and potential applications of cell-specific Zip-Codes in gene delivery and drug targeting in cancer research. |
| first_indexed | 2024-03-07T22:54:53Z |
| format | Article |
| id | doaj.art-01f5fb08798f4da08a8f308c3a5c52f3 |
| institution | Directory Open Access Journal |
| issn | 2234-943X |
| language | English |
| last_indexed | 2024-03-07T22:54:53Z |
| publishDate | 2024-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Oncology |
| spelling | doaj.art-01f5fb08798f4da08a8f308c3a5c52f32024-02-23T04:47:45ZengFrontiers Media S.A.Frontiers in Oncology2234-943X2024-02-011410.3389/fonc.2024.12887241288724A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosisPavan Kumar Puvvula0Lourdes Martinez-Medina1Munevver Cinar2Lei Feng3Andrey Pisarev4Anthony Johnson5Leon Bernal-Mizrachi6Kodikaz Therapeutic Solutions, New York, NY, United StatesKodikaz Therapeutic Solutions, New York, NY, United StatesDepartment of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA, United StatesKodikaz Therapeutic Solutions, New York, NY, United StatesKodikaz Therapeutic Solutions, New York, NY, United StatesKodikaz Therapeutic Solutions, New York, NY, United StatesDepartment of Hematology and Medical Oncology, Winship Cancer Institute of Emory University, Atlanta, GA, United StatesIntroductionWe have demonstrated that transposons derived from ctDNA can be transferred between cancer cells. The present research aimed to investigate the cellular uptake and intracellular trafficking of Multiple Myeloma-zip code (MM-ZC), a cell-specific zip code, in myeloma cell lines. We demonstrated that MM-ZC uptake by myeloma cells was concentration-, time- and cell-type-dependent.MethodsFlow cytometry and confocal microscopy methods were used to identify the level of internalization of the zip codes in MM cells. To screen for the mechanism of internalization, we used multiple inhibitors of endocytosis. These experiments were followed by biotin pulldown and confocal microscopy for validation. Single interference RNA (siRNA) targeting some of the proteins involved in endocytosis was used to validate the role of this pathway in ZC cell internalization.ResultsEndocytosis inhibitors identified that Monensin and Chlorpromazine hydrochloride significantly reduced MM-ZC internalization. These findings suggested that Clathrin-mediated endocytosis and endosomal maturation play a crucial role in the cellular uptake of MM-ZC. Biotin pulldown and confocal microscopic studies revealed the involvement of proteins such as Clathrin, Rab5a, Syntaxin-6, and RCAS1 in facilitating the internalization of MM-ZC. Knockdown of Rab5a and Clathrin proteins reduced cellular uptake of MM-ZC and conclusively demonstrated the involvement of Clathrin-Rab5a pathways in MM-ZC endocytosis. Furthermore, both Rab5a and Clathrin reciprocally affected their association with MM-ZC when we depleted their proteins by siRNAs. Additionally, the loss of Rab5a decreased the Syntaxin-6 association with MMZC but not vice versa. Conversely, MM-ZC treatment enhanced the association between Clathrin and Rab5a.ConclusionOverall, the current study provides valuable insights into the cellular uptake and intracellular trafficking of MM-ZC in myeloma cells. Identifying these mechanisms and molecular players involved in MM-ZC uptake contributes to a better understanding of the delivery and potential applications of cell-specific Zip-Codes in gene delivery and drug targeting in cancer research.https://www.frontiersin.org/articles/10.3389/fonc.2024.1288724/fullmyelomaendocytosisgene therapycancer biologynon-viral vectorscell-specific targeted delivery |
| spellingShingle | Pavan Kumar Puvvula Lourdes Martinez-Medina Munevver Cinar Lei Feng Andrey Pisarev Anthony Johnson Leon Bernal-Mizrachi A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis Frontiers in Oncology myeloma endocytosis gene therapy cancer biology non-viral vectors cell-specific targeted delivery |
| title | A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis |
| title_full | A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis |
| title_fullStr | A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis |
| title_full_unstemmed | A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis |
| title_short | A retrotransposon-derived DNA zip code internalizes myeloma cells through Clathrin-Rab5a-mediated endocytosis |
| title_sort | retrotransposon derived dna zip code internalizes myeloma cells through clathrin rab5a mediated endocytosis |
| topic | myeloma endocytosis gene therapy cancer biology non-viral vectors cell-specific targeted delivery |
| url | https://www.frontiersin.org/articles/10.3389/fonc.2024.1288724/full |
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