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Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

Kirscher L, Deán-Ben XL, Scadeng M, Zaremba A, Zhang Q, Kober C, Fehm TF, Razansky D, Ntziachristos V, Stritzker J, Szalay AA - Theranostics (2015)

Bottom Line: In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models.Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography.At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

View Article: PubMed Central - PubMed

Affiliation: 1. University of Würzburg, Department of Biochemistry, Am Hubland, 97074 Würzburg, Germany.

ABSTRACT
We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

No MeSH data available.


Related in: MedlinePlus

Induction of tyrosinase expression in GLV-1h462 infected A549 cells A) Absorbance measurements at 475nm of cell lysates from rVACV infected A549 cells incubated with different dox concentrations. Significantly higher optical density at 475nm was obtained from GLV-1h462 infected A549 cells at a concentration of 0.05 µg/ml compared to 0.01 µg/ml doxycycline. No significant influence of dox on OD475nm of GLV-1h460, GLV-1h312 or mock infected A549 cancer cells. B) Mean velocity of L-DOPA turnover in A549 cell lysates from cells infected with melanogenic rVACV in presence or absence of dox. Significantly higher velocity of L-DOPA turnover in dox induced than non-induced GLV-1h462 infected A549 cells (p < 0.01). No significant dox-dependent difference in velocity in GLV-1h460 infected cells.
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Figure 2: Induction of tyrosinase expression in GLV-1h462 infected A549 cells A) Absorbance measurements at 475nm of cell lysates from rVACV infected A549 cells incubated with different dox concentrations. Significantly higher optical density at 475nm was obtained from GLV-1h462 infected A549 cells at a concentration of 0.05 µg/ml compared to 0.01 µg/ml doxycycline. No significant influence of dox on OD475nm of GLV-1h460, GLV-1h312 or mock infected A549 cancer cells. B) Mean velocity of L-DOPA turnover in A549 cell lysates from cells infected with melanogenic rVACV in presence or absence of dox. Significantly higher velocity of L-DOPA turnover in dox induced than non-induced GLV-1h462 infected A549 cells (p < 0.01). No significant dox-dependent difference in velocity in GLV-1h460 infected cells.

Mentions: To find the dox concentration at which mTyr expression was induced, GLV-1h462 infected A549 cancer cells were incubated in the presence of increasing dox concentrations (0, 0.01, 0.05, 0.1, 0.5 and 1 µg/ml) (Fig. 2A). Mock infected and GLV-1h312 infected A549 lung carcinoma cells served as a negative control for melanin production and showed low background levels as indicated by optical density measurements at 475nm (OD475). Non-infected A549 cells had an almost constant OD475 of about 0.1 ± 0.01 regardless of the applied dox concentration. The somewhat lower OD475 of GLV-1h312 infected cells compared to mock infected cells can be explained by reduced cell numbers due to viral infection. In case of GLV-1h460 infected A549 cells, which produced melanin in a constitutive manner (Fig. 1A and B), a nearly constant high absorbance of 0.28 ± 0.01 at 475 nm could be detected regardless of the amounts of dox in the medium. In contrast, GLV-1h462 infected A549 cancer cells showed a low optical density at a dox concentration of 0 µg/ml (0.087 ± 0.003) and 0.01 µg/ml (0.103 ± 0.014). At a dox level of 0.05 µg/ml a significant increase (p < 0.001) in OD475 (0.284 ± 0.009), and therefore melanin synthesis, was detected. This dox concentration (0.05 µg/ml) seemed to saturate the inducible system in vitro as no further increase in OD475 with higher dox concentrations was observed.


Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

Kirscher L, Deán-Ben XL, Scadeng M, Zaremba A, Zhang Q, Kober C, Fehm TF, Razansky D, Ntziachristos V, Stritzker J, Szalay AA - Theranostics (2015)

Induction of tyrosinase expression in GLV-1h462 infected A549 cells A) Absorbance measurements at 475nm of cell lysates from rVACV infected A549 cells incubated with different dox concentrations. Significantly higher optical density at 475nm was obtained from GLV-1h462 infected A549 cells at a concentration of 0.05 µg/ml compared to 0.01 µg/ml doxycycline. No significant influence of dox on OD475nm of GLV-1h460, GLV-1h312 or mock infected A549 cancer cells. B) Mean velocity of L-DOPA turnover in A549 cell lysates from cells infected with melanogenic rVACV in presence or absence of dox. Significantly higher velocity of L-DOPA turnover in dox induced than non-induced GLV-1h462 infected A549 cells (p < 0.01). No significant dox-dependent difference in velocity in GLV-1h460 infected cells.
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Related In: Results  -  Collection

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Figure 2: Induction of tyrosinase expression in GLV-1h462 infected A549 cells A) Absorbance measurements at 475nm of cell lysates from rVACV infected A549 cells incubated with different dox concentrations. Significantly higher optical density at 475nm was obtained from GLV-1h462 infected A549 cells at a concentration of 0.05 µg/ml compared to 0.01 µg/ml doxycycline. No significant influence of dox on OD475nm of GLV-1h460, GLV-1h312 or mock infected A549 cancer cells. B) Mean velocity of L-DOPA turnover in A549 cell lysates from cells infected with melanogenic rVACV in presence or absence of dox. Significantly higher velocity of L-DOPA turnover in dox induced than non-induced GLV-1h462 infected A549 cells (p < 0.01). No significant dox-dependent difference in velocity in GLV-1h460 infected cells.
Mentions: To find the dox concentration at which mTyr expression was induced, GLV-1h462 infected A549 cancer cells were incubated in the presence of increasing dox concentrations (0, 0.01, 0.05, 0.1, 0.5 and 1 µg/ml) (Fig. 2A). Mock infected and GLV-1h312 infected A549 lung carcinoma cells served as a negative control for melanin production and showed low background levels as indicated by optical density measurements at 475nm (OD475). Non-infected A549 cells had an almost constant OD475 of about 0.1 ± 0.01 regardless of the applied dox concentration. The somewhat lower OD475 of GLV-1h312 infected cells compared to mock infected cells can be explained by reduced cell numbers due to viral infection. In case of GLV-1h460 infected A549 cells, which produced melanin in a constitutive manner (Fig. 1A and B), a nearly constant high absorbance of 0.28 ± 0.01 at 475 nm could be detected regardless of the amounts of dox in the medium. In contrast, GLV-1h462 infected A549 cancer cells showed a low optical density at a dox concentration of 0 µg/ml (0.087 ± 0.003) and 0.01 µg/ml (0.103 ± 0.014). At a dox level of 0.05 µg/ml a significant increase (p < 0.001) in OD475 (0.284 ± 0.009), and therefore melanin synthesis, was detected. This dox concentration (0.05 µg/ml) seemed to saturate the inducible system in vitro as no further increase in OD475 with higher dox concentrations was observed.

Bottom Line: In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models.Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography.At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

View Article: PubMed Central - PubMed

Affiliation: 1. University of Würzburg, Department of Biochemistry, Am Hubland, 97074 Würzburg, Germany.

ABSTRACT
We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

No MeSH data available.


Related in: MedlinePlus