**3.12 Neuroblastoma tumor treatment with the combination of siRNA-HDAC8 + siRNA-MYC**

We explored the therapeutic effect of the combination of siRNA-HDAC8 + siRNA-MYC treatment on neuroblastoma tumor xenograft in mice. A total of 1x106 mouse neuroblastoma cells in 50% Matrigel were inoculated subcutaneously in 6 weeks old female A/J mice. Tumors were formed with an average

**113**

**Figure 9.**

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells…*

HDAC8 compared to tumors treated with control negative siRNA.

**3.13 The quantitation of targets HDAC 8 and MYC in tumors**

volume of 100 mm3, 12 days after cells were inoculated. A 3 nmol negative control siRNA (NC-siRNA) or a 3 nmol combination of siRNA-HDAC + siRNA-MYC complexed with Lipofectamine RNAi max (Invitrogen) were inoculated into 10 tumors each by intratumoral injections every 3rd day. Tumor growth was measured every 2 days with a caliper and volume was calculated with the formula, length X width2/2. The growth of control tumors treated with NC-siRNA increased, however, the tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC show inhibition of the growth of tumors (**Figure 8A**). The rates of tumor growth were significantly decreased when treated with combined siRNA-MYC + siRNA-

All mice experiments were performed under IACUC approved animal study

Experiment was stopped when the control tumors reached an average volume of over 1200 mm3, then mice were euthanized by CO2 and tumors were removed and weighed. Pictures of mice with tumors were taken before tumors were removed (**Figure 8B**) and pictures of tumors removed and weighed are shown in (**Figure 8C**). The average wet weight of 8 control tumors treated with NC-siRNA and tumors treated with combination of siRNA-HDAC + siRNA-MYC is presented in **Figure 8D**. The average weight of tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC was decreased by 5-fold [0.186 g] compared to average weight of control tumors [1 g] treated with NC-siRNA. Tumor xenograft experiment was repeated twice with 10 mice treated with NC-siRNA and 10 mice treated with a combination of

Tumor targets HDAC 8 and MYC proteins were quantitated by ELISA in extracts

prepared from tumors treated with negative control siRNA and combination of siRNA-HDAC 8 + siRNA-MYC treated tumors. The results indicate that targets HDAC 8 and MYC were decreased by 85% and 65% in tumors treated with the

*(A, B) Quantitation of Myc and Hdac8 proteins from control and combination of siRNA treated tumors. Tumor targets Hdac8 and Myc proteins were quantitated by ELISA in extracts prepared from 3 tumors treated with negative control siRNA and 3 tumors treated with the combination of siRNA-HDAC 8 + siRNA-MYC. Average targets Hdac8 (A) and Myc (B) proteins were decreased by 85% and 65% in tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC compared to tumors treated with NC-siRNA. SEM bars represent the standard deviation from 3 tumors (P\* < 0.030, P\*\* < 0.01). (figures printed from the article* 

*published in "J Cancer Biol Therap," N. Prashad 6(1): 301–307 (2020)).*

*DOI: http://dx.doi.org/10.5772/intechopen.96021*

protocol.

siRNA-HDAC8 + siRNA-MYC.

#### **Figure 8.**

*(A) Effect of combination of siRNA on the growth of neuroblastoma tumor. Murine neuroblastoma cells, 1 × 106 cells in DMEM media with 50% matrigel in 100 ul were subcutaneously injected on the right flanks. After 12 days, tumor growth can be seen and tumors were measured with a caliper. When tumors reached 100 mm3 in size, mice were divided into two groups with 8 mice in each group. Negative control siRNA or combination of siRNA-MYC + siRNA-HDAC8 was mixed with Lipofectamine RNAi max (liposome) in DMEM media without fetal bovine serum and without antibiotic. siRNA complexed with Lipofectamine in a volume of 30 ul was delivered into tumors by intratumoral injection every third day. Tumors were measured every second day with a caliper and mice were weighed every third day. Tumor volume was calculator with a formula, V = length × width 2 /2. The numbers on X-axis show the number of siRNA treatments. Control tumor growth is shown by diamond (^) markers and the growth of tumors treated with siRNAs is shown by round (0) markers. SEM bars represent the standard deviation from 8 mice at each point P\* < 0.031. (figure is printed from published article in "J. Cancer Biology and Therapeutics" N. Prashad V 6: 301–307 2020). (B) Mice with control and combination of siRNA treated tumors. Top row of mice with control tumors treated with NC siRNA and the bottom row of mice with tumors treated with the combination of siRNA-MYC + siRNA-HDAC. (C) Tumors from control and combination of siRNA treated mice. Top row representative control tumors treated with NC-siRNA and the bottom row representative tumors treated with the combination of siRNA-MYC + siRNA-HDAC8. (figure is printed from published article in "J. Cancer biology and therapeutics" N. Prashad V 6: 301–307 2020). (D) Weights of control and combination of siRNA treated tumors. Average of 8 control tumors was 1 gram and average of 8 tumors treated with combination of siRNA MYC + siRNA-HDAC8 was 0.186 gram. SEM bars represent the standard deviation from 8 tumors P\*\* < 0.004.*

#### *Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells… DOI: http://dx.doi.org/10.5772/intechopen.96021*

volume of 100 mm3, 12 days after cells were inoculated. A 3 nmol negative control siRNA (NC-siRNA) or a 3 nmol combination of siRNA-HDAC + siRNA-MYC complexed with Lipofectamine RNAi max (Invitrogen) were inoculated into 10 tumors each by intratumoral injections every 3rd day. Tumor growth was measured every 2 days with a caliper and volume was calculated with the formula, length X width2/2. The growth of control tumors treated with NC-siRNA increased, however, the tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC show inhibition of the growth of tumors (**Figure 8A**). The rates of tumor growth were significantly decreased when treated with combined siRNA-MYC + siRNA-HDAC8 compared to tumors treated with control negative siRNA.

All mice experiments were performed under IACUC approved animal study protocol.

Experiment was stopped when the control tumors reached an average volume of over 1200 mm3, then mice were euthanized by CO2 and tumors were removed and weighed. Pictures of mice with tumors were taken before tumors were removed (**Figure 8B**) and pictures of tumors removed and weighed are shown in (**Figure 8C**). The average wet weight of 8 control tumors treated with NC-siRNA and tumors treated with combination of siRNA-HDAC + siRNA-MYC is presented in **Figure 8D**. The average weight of tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC was decreased by 5-fold [0.186 g] compared to average weight of control tumors [1 g] treated with NC-siRNA. Tumor xenograft experiment was repeated twice with 10 mice treated with NC-siRNA and 10 mice treated with a combination of siRNA-HDAC8 + siRNA-MYC.

### **3.13 The quantitation of targets HDAC 8 and MYC in tumors**

Tumor targets HDAC 8 and MYC proteins were quantitated by ELISA in extracts prepared from tumors treated with negative control siRNA and combination of siRNA-HDAC 8 + siRNA-MYC treated tumors. The results indicate that targets HDAC 8 and MYC were decreased by 85% and 65% in tumors treated with the

#### **Figure 9.**

*Pheochromocytoma, Paraganglioma and Neuroblastoma*

**siRNA-HDAC8 + siRNA-MYC**

**3.12 Neuroblastoma tumor treatment with the combination of** 

We explored the therapeutic effect of the combination of siRNA-

HDAC8 + siRNA-MYC treatment on neuroblastoma tumor xenograft in mice. A total of 1x106 mouse neuroblastoma cells in 50% Matrigel were inoculated subcutaneously in 6 weeks old female A/J mice. Tumors were formed with an average

**112**

*represent the standard deviation from 8 tumors P\*\* < 0.004.*

**Figure 8.**

*(A) Effect of combination of siRNA on the growth of neuroblastoma tumor. Murine neuroblastoma cells, 1 × 106 cells in DMEM media with 50% matrigel in 100 ul were subcutaneously injected on the right flanks. After 12 days, tumor growth can be seen and tumors were measured with a caliper. When tumors reached 100 mm3 in size, mice were divided into two groups with 8 mice in each group. Negative control siRNA or combination of siRNA-MYC + siRNA-HDAC8 was mixed with Lipofectamine RNAi max (liposome) in DMEM media without fetal bovine serum and without antibiotic. siRNA complexed with Lipofectamine in a volume of 30 ul was delivered into tumors by intratumoral injection every third day. Tumors were measured every second day with a caliper and mice were weighed every third day. Tumor volume was calculator with a formula, V = length × width 2 /2. The numbers on X-axis show the number of siRNA treatments. Control tumor growth is shown by diamond (^) markers and the growth of tumors treated with siRNAs is shown by round (0) markers. SEM bars represent the standard deviation from 8 mice at each point P\* < 0.031. (figure is printed from published article in "J. Cancer Biology and Therapeutics" N. Prashad V 6: 301–307 2020). (B) Mice with control and combination of siRNA treated tumors. Top row of mice with control tumors treated with NC siRNA and the bottom row of mice with tumors treated with the combination of siRNA-MYC + siRNA-HDAC. (C) Tumors from control and combination of siRNA treated mice. Top row representative control tumors treated with NC-siRNA and the bottom row representative tumors treated with the combination of siRNA-MYC + siRNA-HDAC8. (figure is printed from published article in "J. Cancer biology and therapeutics" N. Prashad V 6: 301–307 2020). (D) Weights of control and combination of siRNA treated tumors. Average of 8 control tumors was 1 gram and average of 8 tumors treated with combination of siRNA MYC + siRNA-HDAC8 was 0.186 gram. SEM bars* 

*(A, B) Quantitation of Myc and Hdac8 proteins from control and combination of siRNA treated tumors. Tumor targets Hdac8 and Myc proteins were quantitated by ELISA in extracts prepared from 3 tumors treated with negative control siRNA and 3 tumors treated with the combination of siRNA-HDAC 8 + siRNA-MYC. Average targets Hdac8 (A) and Myc (B) proteins were decreased by 85% and 65% in tumors treated with the combination of siRNA-HDAC8 + siRNA-MYC compared to tumors treated with NC-siRNA. SEM bars represent the standard deviation from 3 tumors (P\* < 0.030, P\*\* < 0.01). (figures printed from the article published in "J Cancer Biol Therap," N. Prashad 6(1): 301–307 (2020)).*

combination of siRNA-HDAC8 + siRNA-MYC compared to tumors treated with NC-siRNA (**Figure 9A** and **B**).

The results indicate that a decrease in the tumor targets HDAC 8 and MYC caused the inhibition of the growth of tumors.

#### **4. Discussion**

miRNAs are both oncogenic and tumor suppressors. In normal cells homeostasis is maintained by keeping equilibrium between oncogenic and suppressor miRNA. If this equilibrium is disrupted that can cause dysfunction with increases in oncogenic miRNA and decreases in suppressor miRNA. A decrease in a specific suppressor miRNA can cause overexpression of HDCAs, c MYC and MYCN which can alter gene expression and cause cancer. However, when suppressor miRNAs are added exogenously to these cells then these cells restore normal properties and show growth arrest and apoptosis Therefore, suppressor miRNAs seem to be critical in the maintenance of cellular homeostasis.

A decrease in suppressor miRNA can over express genes like c MYC, MYCN and HDACs and cause cancers. Over expression of c MYC and MYCN cause the down regulation of suppressor miRNAs. HDACs indirectly effect gene expression by the deacetylation of histones, therefore, this process can also effect the expression of miRNA.

Transfection of miR-665 into murine NB cells caused growth inhibition, cell cycle arrest, decreased total HDAC activity, decreased HDAC8 and MYC protein expression, activated caspase 3 and increased the acetylation of histones. miR-665 targets HDAC8, c MYC and MYCN oncogene and decreases their expression. These targets are validated by the co- transfection of luciferase reporter with target 3' UTR and miRNA-665. Therefore, miRNA 665 directly targets HDAC 8, MYCN and c MYC in the inhibition of mouse neuroblastoma cells. This is the first report to show that miR-665 is a suppressor miRNA of mouse neuroblastoma.

In targeted therapy of cancer, critical genes and proteins involved in the tumorigenesis are identified and therapeutic agents' miRNA and siRNA are used to inhibit the expression of target genes to inhibit the growth of cells in vitro and in vivo. SiRNA-mediated gene knockdown is much more potent and specific with only one mRNA target, whereas miRNA has multiple mRNA targets. siRNA therapeutic approach was used in gene targeting overexpressed cancer proteins in inhibiting cancer cell growth in vitro and inhibited tumor growth in vivo in the following mouse models: breast cancer mouse model, Glioma cells tumor and colon cancer tumor. MYCN, c-MYC, and HDAC8 may each contribute to neuroblastoma tumorigenesis. We reported that transfection of mimic suppressor miR-665 inhibited the expression of c-MYC and HDAC 8 and increased caspase 3 involved in apoptosis and inhibited the growth of neuroblastoma cells in vitro [10].

Our data also indicate that both c-MYC and HDAC 8 are critical targets and targeting these two targets with siRNA inhibited cell growth by 86% in vitro. The combination of siRNAs inhibited tumor growth in vivo by 80%, therefore, inhibiting more than one target is critical for the successful treatment of tumors in vivo.

#### **5. Conclusion**

Neuroblastoma is the most frequently diagnosed extracranial solid tumor in children. These tumors account for 15% of childhood deaths from cancer. Survival in one- year-old children is <30% despite aggressive therapies.

**115**

**Author details**

Nagindra Prashad

Genetics in Medicine, Houston, TX, USA

provided the original work is properly cited.

\*Address all correspondence to: nagindra.prashad@gmail.com

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells…*

We used mouse neuroblastoma tumor model and identified MYC and HDAC8 are the critical targets in neuroblastoma tumorigenesis. Treatment of the tumors in mice with the combination of siRNA-MYC + siRNA-HDAC8 inhibited both the targets MYC and HDAC8 simultaneously and inhibited the growth of tumor by 80%. Therefore, inhibiting more than one target is critical for the successful treatment of

The author would like to thank Texas Children's Hospital Flow Cytometry Core Facilities, Houston, Texas, for their help in performing cell cycle analysis and Dr. George Calin and associates at MD Anderson Cancer Center, Houston, Texas, for

Ethics approval: Mice experiments were performed with the approval of the institutional Animal Care and Use Committee, IACUC at Nanospectra Biosciences

TMC Internist, Houston, Texas provided partial funding for the chemical sup-

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

The author declares that they have no conflicts of interest.

*DOI: http://dx.doi.org/10.5772/intechopen.96021*

access to the luminometer plate reader.

tumors in vivo.

**Acknowledgements**

**Conflicts of interest**

**Declarations**

Inc. Houston, Texas.

plies used in this project.

**Grant support**

*Targeting MYC and HDAC8 with a Combination of siRNAs Inhibits Neuroblastoma Cells… DOI: http://dx.doi.org/10.5772/intechopen.96021*

We used mouse neuroblastoma tumor model and identified MYC and HDAC8 are the critical targets in neuroblastoma tumorigenesis. Treatment of the tumors in mice with the combination of siRNA-MYC + siRNA-HDAC8 inhibited both the targets MYC and HDAC8 simultaneously and inhibited the growth of tumor by 80%. Therefore, inhibiting more than one target is critical for the successful treatment of tumors in vivo.
